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https://openalex.org/W2788766865	https://nottingham-repository.worktribe.com/preview/922338/LETIS%20Submission%20Final%20Accepted%20Version.pdf	English	null	Insights Into the Different Effects of Food on Intestinal Secretion Using Magnetic Resonance Imaging	JPEN, Journal of parenteral and enteral nutrition/JPEN. Journal of parenteral and enteral nutrition	2,018	cc-by	4,868	These authors assert joint first authorship These authors assert joint first authorship 1 1 1 Author Names: Victoria C Wilkinson-Smith, BMBS1,2, Giles Major, PhD, MRCP1,2 , Lucy Ashleigh 2 , Kathryn Murray, PhD 1,3, Caroline L Hoad, PhD1,3 , Luca Marciani, PhD 1,2,3, Penny A Gowland, PhD 1,3, Robin C Spiller, MD, FRCP 1,2 and the Nottingham GI MRI research group. Title Page Title Insights into the effects of food on intestinal secretion using magnetic resonance imaging Author Affiliations 1. National Institute for Health Research (NIHR) Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust and University of Nottingham. (VWS, GM, KM, CH, LM, PG, RS) 2. Nottingham Digestive Diseases Centre, University of Nottingham, Nottingham, United Kingdom 2. Nottingham Digestive Diseases Centre, University of Nottingham, Nottingham, United Kingdom (VWS, GM, LA, LM, RS) 3. Sir Peter Mansfield Imaging Centre, University of Nottingham, Nottingham, United Kingdom 3. Sir Peter Mansfield Imaging Centre, University of Nottingham, Nottingham, United Kingdom Disclaimers 2 2 None Corresponding Author Professor Robin Spiller Mailing Address: QMC West Block E Floor Queen's Medical Centre Nottingham NG7 2UH UK Email: robin.spiller@nottingham.ac.uk Telephone: 0115 823 1090 Sources of Support Funded by the University of Nottingham Short running head: Lettuce, Bread & Rhubarb Impact on Bow Abbreviations MRI (Magnetic Resonance Imaging) SBWC (Small bowel water content) ACWC (Ascending colon water content) Abbreviations 3 3 FODMAPs (fermentable oligo-, di-, mono-saccharides and polyols) AQP3 (aquaporin 3) Design Crossover trial of three isocaloric meals in healthy adults. Subjects underwent MRI scans fasting and hourly to 180min postprandial. Meals: 2 slices white bread with 10g butter; 300g rhubarb with 60mL lactose-free cream; 300g lettuce with 30mL mayonnaise. Visits one week apart. Primary outcome: small bowel water content (SBWC) using MRI. Secondary outcomes: ascending colon water content; T1 relaxation time of ascending colon (T1AC); gastric volume; visual analogue scales of bloating and satiety (0-100) scored half-hourly. Background Ileostomy output determines need for parenteral fluid support. Plant foods may contain chemicals that stimulate intestinal secretion eg. lactucins in lettuce and rhein in rhubarb. These may account for increases in ileostomy output but experimental demonstration of this is burdensome for patients. Clinical Trial Registration: Registered on clinicaltrials.gov, October 13, 2016. Reference: NCT02939716 4 4 Objective Determine the effect of different foods on intestinal water content in healthy volunteers using Magnetic Resonance Imaging (MRI). Results 5 subjects completed the study. 9 female, 6 male, median age 21(IQR 20,22). 15 subjects completed the study. 9 female, 6 male, median age 21(IQR 20,22). Bread induced a fall in SBWC compared to a rise after lettuce and greater rise after rhubarb, with significant differences in the area under the curve (0-3 hours) of the change from baseline (P<0.01, paired t-test). Rhubarb increased T1AC but 5 5 differences at 3 hours were not significant (P=0.06). Symptom scores were higher for lettuce >rhubarb >bread. differences at 3 hours were not significant (P=0.06). Symptom scores were higher for lettuce >rhubarb >bread. Keywords MRI, intestinal failure, ileostomy, short bowel syndrome, lactucins, rhein Conclusion Lettuce and rhubarb meals increased intestinal water content, demonstrating how different foods can alter small bowel secretion. MRI can be used to evaluate the effects of diet, enteral feed and pharmaceuticals on intestinal content and function. Clinical Relevancy Statement This paper shows a novel MRI technique for assessment of the secretory effects of foods. This could be applied to study a range of different formulations designed to reduce ileostomy or jejunostomy output in patients with the short bowel syndrome. 6 6 Introduction In patients with a small bowel stoma, high stoma output impairs quality of life and leads to dehydration and in extreme cases induces acute kidney injury. High stoma output is commonly caused by an inadequate length of small bowel remaining after resection to maintain absorptive function (short bowel) although partial small bowel obstruction, infection and inflammation can also contribute. Chronic high output is the most significant factor in determining the need for parenteral nutritional support. There is a limited evidence base for interventions to reduce stoma output. Agents such as loperamide, codeine which slow transit and proton pump inhibitors to reduce gastric secretions are recommended. A glucagon-like peptide 2 analogue (teduglutide) has also shown some benefit, possibly by stimulating mucosal growth and enhancing nutrient absorption1. Modifying dietary intake also plays an important role particularly sodium intake. Patients are currently advised to restrict or avoid drinking oral hypotonic fluids, as these can cause further loss of water and sodium, and instead drink an oral sodium and glucose solution. Composition of dietary intake is also important; diets that are high in fermentable oligo-, di-, mono-saccharides and polyols (FODMAPs) have been shown to increase ileostomy output by 22%2, demonstrating that the osmotic effects of food can alter the balance between secretion and absorption in the small bowel. Other mechanisms including stimulation of secretion may also operate. A survey of ileostomy patients looking at a large variety of different foodstuffs found that both rhubarb and lettuce were perceived to increase stoma output3 though this was not objectively confirmed. Objective experimental quantification of such effects is problematic because of patient heterogeneity and also because such studies Composition of dietary intake is also important; diets that are high in fermentable oligo-, di-, mono-saccharides and polyols (FODMAPs) have been shown to increase ileostomy output by 22%2, demonstrating that the osmotic effects of food can alter the balance between secretion and absorption in the small bowel. Other mechanisms including stimulation of secretion may also operate. A survey of ileostomy patients looking at a large variety of different foodstuffs found that both rhubarb and lettuce were perceived to increase stoma output3 though this was not objectively confirmed. Objective experimental quantification of such effects is problematic because of patient heterogeneity and also because such studies 7 imposes a burden on frail patients who have significant health problems. This limits its suitability for early phase assessment of therapeutic interventions. Study design The study was a three-period, three-treatment crossover design with randomisation of treatment order. The protocol was approved by the Ethics Committee of the University of Nottingham and volunteers gave written informed consent. The study was carried out according to Good Clinical Practice as defined by the Declaration of Helsinki. The study was registered on clinicaltrials.gov, reference: NCT02939716. Introduction We have validated techniques to measure free water in the intestine4 and assess the physico-chemical composition of the colonic chyme5 and have previously shown that bread, which is rapidly digested to free monomeric sugars and absorbed, leads to a reduction in small bowel water6. Our aim was to apply these techniques to investigate the effects of equicaloric lettuce and rhubarb meals on intestinal fluid distribution, in comparison with a standard bread meal. We hypothesised that rhubarb and lettuce would both significantly increase small bowel water when compared with bread, and that, in healthy subjects with an intact colon, we would be able to detect a consequent increased fluidity of the ascending colon content. Study participants Subjects were aged ≥18 and able to give consent. Subjects were excluded if they had a) history of GI surgery (other than appendicectomy), GI disorder, or a positive 8 diagnosis of irritable bowel syndrome by Rome III criteria b) contra-indication to MRI or any medical condition compromising participation, such as an inability to lie in the MRI scanner c) inability to cease use of medication affecting gut function or follow the dietary restrictions required for the study d) lifestyle factors likely to disrupt gut function or compliance with the protocol such as night shift working. All subjects gave written consent. Test meals Three isocaloric test meals were used: 1) 2 slices white bread (Tesco Stores Ltd., Welwyn Garden City, U.K.) with 10g butter (Lurpak, Arla Foods Ltd, Leeds, UK) (10g fat, 248 kcal); 2) 300g organically grown rhubarb, cooked by microwave, with 60mL lactose-free cream (Arla Foods, Leeds, UK) and 2 sweetener tablets (Hermesetas, Hermes Sweeteners Ltd, Zurich.) (22.5g fat, 243 kcal); 3) 300g Romaine lettuce (Tesco Stores Ltd., Welwyn Garden City, U.K.) with 30mL mayonnaise (Hellman’s, Unilever, London, UK) (23.5g fat, 246 kcal). Each of these was consumed with 200ml water. Subjects were required to consume all of the meal and water. Interventions and Procedures Consent, screening and enrolment took place at the Nottingham Digestive Diseases Biomedical Research Unit, Nottingham. All subsequent study visits took place at the 1.5T scanner annex of the Sir Peter Mansfield Imaging Centre, Nottingham. Subjects visited the Centre on three separate occasions, with at least one week between each visit to minimise any carryover effect. Subjects were asked to eat similar foods in the 24 hours before each visit and to avoid apples, beans, peas, pulses, sweetcorn, dietary supplements and alcohol. They abstained from caffeine and strenuous exercise for 18 hours before the visit and did not eat after 8pm on the evening beforehand. On the day of the visit subjects fasted before attendance, other than sips of water with essential medicines. On arrival they completed two 0-100 visual analogue scales to assess bloating and satiety, and underwent an MRI scan. They were then given 15 minutes to consume the test meal before repeat assessment of symptoms and further MRIs (t=0 min). Symptom scores were repeated at 30 minute intervals for 9 9 180 minutes on the same 0-100mm visual analogue scales. MRIs were repeated every 60 minutes for 180 minutes. 180 minutes on the same 0-100mm visual analogue scales. MRIs were repeated every 60 minutes for 180 minutes. Outcomes The primary outcome was small bowel water content (SBWC, mL). The technique measures the volume of freely mobile water using a previously validated MRI technique. Interventions were compared for the area under the curve [AUC, 0-180 minutes] of the change from fasting baseline. Secondary MRI outcomes were: ascending colon water content (ACWC, mL); This is measured in the same way as the small bowel water content and reflects only freely mobile water with long transverse relaxation time, and is only a small fraction of the total water7. We therefore also measured ascending colon longitudinal relaxation time (T1AC, ms) which in this context can provide a more useful assessment of colonic contents. , T1 is a fundamental MRI parameter that reflects the time constant for the water 10 hydrogen protons to return to their equilibrium state following a radiofrequency excitation pulse. In the context of this paper, more watery chyme has a longer T1 relaxation time. Gastric volume (mL) was measured to identify any differences in gastric emptying. Symptom scores for bloating and satiety were also compared. MRI protocol MR scanning was performed on a whole-body 1.5T scanner (Achieva, Philips Medical System, Best, The Netherlands). Volunteers were positioned feet first and supine in the scanner with a 16-element SENSE (Philips Medical System) receive torso coil around their abdomen. The SBWC and ACWC were measured using a single shot, fast spin echo sequence (rapid acquisition with relaxation enhancement, RARE) to acquire in a single breath- hold 24 coronal images with in-plane resolution interpolated to 0.78 mm x 0.78 mm and a slice thickness of 7 mm, with no gap between slices (TR = 8000 ms, TEeff = 320 ms, AQR = 1.56 mm x 2.90 mm) as previously described (11). This sequence yields high intensity signals from areas with fluid and little signal from body tissues. T1AC was measured using a single slice balanced turbo field echo (bTFE) sequence with a preparatory 180 degrees inversion pulse applied before acquiring the imaging data. Data was acquired from 8 different inversion times (TI) (time between inversion pulse and imaging pulses) ranging from 25 - 4925 ms. 11 Gastric volumes were measured using a balanced gradient echo (called balanced turbo field echo, bTFE or trueFISP) sequence (TR = 3.0 ms, TE = 1.5 ms, FA 80°, SENSE factor 2.0) to acquire 50 transverse images each with an in-plane resolution of 2.00 mm x 1.77 mm and slice thickness of 5 mm, with no gap between slices. Images were acquired during an expiration breath hold lasting between 13-24 seconds. Each period of imaging lasted between 10 and 15 min, after which volunteers were removed from the scanner and allowed to sit upright. Randomisation and Blinding Randomisation of treatment order was achieved using the remote, online programme www.randomization.com. Neither subjects nor researchers were blind to the meal consumed on study days. Scans were coded and pseudo-anonymised through a shell programme so that MRI analysis was conducted blind to the intervention. Results Recruitment took place from September 2016 to January 2017. 18 subjects were enrolled, of whom 3 were unable to complete the study due to scanner technical problems (Figure 1). 15 subjects completed the study: 9 female, 6 male; median age 21 (IQR 20, 22) and mean body mass index 21.4 kg.m-2 (SD ±2.2). Power and statistical analysis This was a pilot study as no data are available on the effect of ingested lettuce or rhubarb on MRI parameters. However using n=16 we had previously detected significant differences in small bowel water content induced by the mild laxative bran7 which were less than the changes anticipated in this study. 12 Statistical tests were performed using GraphPad Prism version 7.03 for Windows, (GraphPad Software, La Jolla California USA). Data normality was assessed using Shapiro–Wilk test. Two-way analysis of variance was used to assess for any difference between the test meals during the postprandial period. Any difference identified was then explored by direct comparison between meals. The Wilcoxon Signed Rank test for non-parametric paired data was used. Tukey’s multiple comparisons test was performed to adjust for multiple comparisons. Statistical differences were considered significant at P<0.05. Small bowel water content (SBWC) There was no difference in mean fasting SBWC between intervention days but responses to the meal varied considerably (Table 1, Figure 2). The AUC [0-180 mins] of the change from baseline was significantly different between all interventions (P <0.01 for each paired t-test). Bread induced a fall in SBWC similar to that seen in previous studies (14), from fasting mean 4728 mL to 44 mL at 60 mins (P <0.001). No such decline was seen with lettuce or rhubarb. Mean SBWC with the lettuce meal was 6055 mL at baseline and 6553 mL at 60 minutes, rising 13 significantly at 180 mins to 10850 mL (P <0.005). With the rhubarb meal, mean SBWC increased after 60 mins, from 5533 mL at fasting to 10344 mL (P <0.001), then 14161 mL and 142 45mL at 120 and 180 mins respectively, a peak that was significantly higher than for lettuce (P <0.05). Gastric Volumes As expected, given the energy density and corresponding size of the meal, gastric volumes were similar immediately after rhubarb and lettuce (55889mls and 57192 mls respectively), in both cases significantly larger than bread (314108 mls) (P<0.001). AUC and peaks are shown in Table 3. Gastric half-emptying times were calculated from the volumes data. Lettuce emptied fastest (6936 mins), followed by rhubarb (8024 mins) then bread (10435 mins). This difference was significant for lettuce vs. bread (p =0.014) but not for lettuce vs. rhubarb (p = 0.62) or bread vs. rhubarb (p = 0.136). Ascending colon free water content and relaxation times Mean values for ascending colon (freely mobile) water content (ACWC) are shown in Figure 3, with AUC and peak shown in Table 3. As previously reported with standard rice meals, volumes were low; during the bread intervention only one subject had >1 mL detectable at any point. After lettuce, four subjects had detectable ACWC on the immediate post-prandial scan, with volumes of 3, 12, 12, and 45 mL but by 180 mins it was only detectable in two subjects with 2 and 4 mL respectively. Significantly more ACWC was detectable after rhubarb than bread (P <0.01 Wilcoxon) as by 180 mins seven subjects had >1 mL detectable. The fact than many subjects had no free water detectable is in keeping with previous work and reflects the fact that freely mobile water entering the colon from the ileum is rapidly absorbed or mixed with the chyme thereby losing its MR signal. In contrast, relaxation times (T1), which are a marker of the fluidity of the colonic chyme that does not depend exclusively on free water, could be readily measured in all subjects and rose after all meals. Absolute values are shown in Table 2 with change from baseline shown in Figure 4. The changes after bread and lettuce were similar with mean rise of 111206 ms and 143262ms respectively. Rhubarb induced a larger 14 rise of 313318 ms but the difference from bread just failed to reach conventional statistical significance (mean difference 202 ms, 95% CI -12 to 416, P =0.06). rise of 313318 ms but the difference from bread just failed to reach conventional statistical significance (mean difference 202 ms, 95% CI -12 to 416, P =0.06). Bloating and Satiety Both bloating and satiety scores (0 - 100 on VAS) peaked immediately after the meal (t= 0mins) for all three meals. For bloating, scores were highest for lettuce (4523) then rhubarb (3320) then bread (2814). Scores for bread vs. lettuce remained significantly different up to t= 90mins (p= 0.036). Peak satiety scores for lettuce (6419) were significantly higher than for both rhubarb (4713, p= 0.01) and bread 4920 (p = 0.03). AUC and peaks for these symptoms are shown in Table 3 15 Discussion The aim of the study was to use non-invasive methods to characterise and quantify intestinal fluid distribution after different meals. This is the first study to use MRI to address the effect of foods specifically implicated in affecting stoma output. Our hypothesis, that lettuce and rhubarb would increase SBWC compared to bread, was confirmed, with differential effects also found between the two test meals. This objective demonstration of a physiological effect supports the subjective reports of ileostomy patients that these foods stimulate increased stoma outflow. The fall in SBWC after the bread meal is similar to our previous studies6 and likely reflects the very rapid hydrolysis of wheat starch by superabundant amylase in the proximal duodenum. The liberated glucose is rapidly absorbed, and with it water, so that both the blood glucose profile, which in other studies peaks within 30 minutes8, and SBWC profile, mimic those seen with a pure glucose solution9. In contrast, the striking rise in SBWC after the rhubarb and the less marked, but still significant, rise after lettuce may reflect several different processes. Both meals had considerable fat content and some of the increase in SBWC will be due to pancreatico-biliary secretions, stimulated by a cholecystokinin response to hydrolysed fat in the duodenum. Previous studies in Nottingham have shown that high fat meals induce marked increases in SBWC, an effect enhanced by using a stable emulsion10 such as mayonnaise. 16 The rhubarb and lettuce meals were matched for fat content so the difference between these meals is likely to reflect other factors present in those foods. Rhubarb has been used for many years as a laxative agent in the practice of traditional Chinese/herbal medicine11. This laxative effect is caused by the compounds rheinanthrone and rhein which account for 2% by weight of rhubarb and appears to act similarly to bisacodyl by activating macrophages, increasing PGE2 and thereby decreasing colonic expression of AQPs12. Lettuce is another food that was reported to increase stoma output by 1 in 6 patients3 but is less commonly associated with laxative effects in an intact bowel. Some lettuce varieties exude a milk-like latex material (Lactucarium) when cut. This is a mild cytotoxin13 and so could be expected to stimulate intestinal secretion. Discussion Having considered direct chemical stimulation by food components, it should also be noted that the fibrous nature of rhubarb and lettuce may lead to partial obstruction in ileostomy patients, stimulating increased secretion. Such an effect cannot be excluded in our volunteers although abdominal pain, nausea and vomiting were not reported during the study. Although the exact mechanism of such effects remains uncertain, the observed alterations in SBWC will not necessarily alter bowel function in a subject with an intact colon; the colon can normally compensate for the wide variation in fluid 17 17 entering from the ileum that can be expected from a normal mixed diet. As such, little free water is normally found in the resting colon. However we did find some evidence of increased ascending colon water content 180 minutes after rhubarb. The timing of the increase in water content would fit with the first arrival of meal residue in the caecum. The T1 longitudinal relaxation time reflects the freedom of molecules to move relative to each other in a material. We have previously shown that in a study with ispaghula it correlates with stool water content 14(unpublished data). The T1AC results for rhubarb, whilst not reaching significance in this study, also support the arrival of more water/watery contents into the ascending colon. The use of such techniques is a relatively new approach but provides a useful platform to demonstrate proof of principle for therapeutic agents before their application in patients with functional bowel disorders as well as those with high stoma output. We have previously found that, when small bowel water content of volunteers with an intact bowel was increased 400% by 40g of fructose, small bowel diameter only increased by 20%15 suggesting that the small bowel copes with increased volumes by utilising more of the normally collapsed distal bowel rather than by increasing its diameter. This reserve capacity will be limited in patients who have had small bowel resected and will be proportionate to the remaining bowel. This phenomenon would also tend to exacerbate the effect of foods promoting secretion over absorption. 18 Limitation of the study include the small sample size and the 60 minute interval between MRI scans, which limited our ability to accurately define the time course of the changes we observed. Discussion Although the meals were equicaloric comparison between the bread meal and other meals is somewhat limited by differences in meal size, energy density and fat content. Future studies would need to control for these aspects or else use the possible active ingredients in purified form such as Rhein and Lactucarium. Finally, we studied volunteers with intact bowels, with small bowel and colon in continuity. Whilst we might interpret changes in the consistency of chyme in the ascending colon as corresponding to changes in stoma output, this has never been shown. Patients with short bowel syndrome and stomas have different physiology and are likely to have different secretions and transit due to reduced levels of gut hormones such as GLP-1, GLP-2 and PPY. Our study represents a model for investigation but extrapolation to patients’ responses must be qualified and requires further studies in patients. In conclusion, the study has shown that different foods can alter intestinal fluid balance, which may be relevant to dietary management of patients with small bowel stomas. The methods demonstrated are readily applicable to other dietary interventions, either food groups or artificial enteral feeds. Future work should explore the effect of other dietary and pharmaceutical therapies, including food choices, on intestinal fluid balance, and should include correlation between MRI findings and stoma output in patients. The insights gained can then be tested through clinical trials in order to improve treatment recommendations, and reduce the health burden relating to high stoma output. 19 Acknowledgments R. Spiller, G. Major, P. Gowland, L. Marciani, C. Hoad contributed to the conception and design of the research; L. Ashleigh, G. Major, VW. Smith, K. Murray contributed to the acquisition and analysis of the data; L. Ashleigh, K. Murray, G. Major, VW. Smith contributed to the interpretation of the data; VW. Smith, G. Major, C. Hoad, R. Spiller drafted the manuscript; All authors critically revised the manuscript, agree to be fully accountable for ensuring the integrity and accuracy of the work, and read and approved the final manuscript. R. Spiller has received research funding from Norgine and Zespri. He has also acted on advisory boards for Allergan, Commonwealth Diagnostics International, Napo Pharmaceuticals, Ipsen, and Yuhan, and received speakers’ fees from Menarini and Alfawasserman. G. Major has received speaker’s fees from Allergan and Vertex. The remaining authors have no COI to declare. 20 References 1. Nightingale J, Woodward JM. Guidelines for management of patients with a short bowel. Gut. 2006;55(Suppl 4):iv1-iv12. 2. Barrett JS, Gearry RB, Muir JG, et al. Dietary poorly absorbed, short-chain carbohydrates increase delivery of water and fermentable substrates to the proximal colon. Aliment Pharmacol Ther. 2010;31(8):874-882. 3. Thomson TJ, Runcie J, Khan A. The effect of diet on ileostomy function. Gut. 1970;11(6):482-485. 4. Hoad CL, Marciani L, Foley S, et al. Non-invasive quantification of small bowel water content by MRI: a validation study. Phys Med Biol. 2007;52(23):6909-6922. 5. Marciani L, Garsed KC, Hoad CL, et al. Stimulation of colonic motility by oral PEG electrolyte bowel preparation assessed by MRI: comparison of split vs single dose. Neurogastroenterol Motil. 2014;26(10):1426-1436. 6. Marciani L, Pritchard SE, Hellier-Woods C, et al. Delayed gastric emptying and reduced postprandial small bowel water content of equicaloric whole meal bread versus rice meals in healthy subjects: novel MRI insights. Eur J Clin Nutr. 2013;67(7):754-758. 7. Murray K, Hoad CL, Mudie DM, et al. Magnetic Resonance Imaging Quantification of Fasted State Colonic Liquid Pockets in Healthy Humans. Mol Pharm. 2017;14(8):2629-2638. 8. Kristensen M, Jensen MG, Riboldi G, et al. Wholegrain vs. refined wheat bread and pasta. Effect on postprandial glycemia, appetite, and subsequent 21 ad libitum energy intake in young healthy adults. Appetite. 2010;54(1):163- 169. 9. Marciani L, Cox EF, Hoad CL, et al. Postprandial changes in small bowel water content in healthy subjects and patients with irritable bowel syndrome. Gastroenterology. 2010;138(2):469-477, 477 e461. 10. Hussein MO, Hoad CL, Wright J, et al. Fat emulsion intragastric stability and droplet size modulate gastrointestinal responses and subsequent food intake in young adults. J Nutr. 2015;145(6):1170-1177. 11. Barceloux DG. Medical toxicology of natural substances : foods, fungi, medicinal herbs, plants, and venomous animals. Hoboken, N.J.: John Wiley & Sons; 2008. 12. Ikarashi N, Baba K, Ushiki T, et al. The laxative effect of bisacodyl is attributable to decreased aquaporin-3 expression in the colon induced by increased PGE2 secretion from macrophages. Am J Physiol Gastrointest Liver Physiol. 2011;301(5):G887-895. 13. Besharat S, Besharat M, Jabbari A. Wild lettuce (Lactuca virosa) toxicity. BMJ Case Rep. 2009;2009:bcr06.2008.0134. 14. Major G, Murray K, Nowak A, et al. OC-068 Measuring the Effect of Ispaghula on Gut Content and Function Using MRI. Gut. 2016;65(Suppl 1):A40-A41. 15. Murray K, Wilkinson-Smith V, Hoad C, et al. References Differential Effects of FODMAPs (Fermentable Oligo-, Di-, Mono-Saccharides and Polyols) on Small and Large Intestinal Contents in Healthy Subjects Shown by MRI. Am J Gastroenterol. 2014;109(1):110-119. 22 Tables Table 1 – Fasting and postprandial Small Bowel Water Content (SBWC) in mL for the 3 test meals Mean (SEM) Fasting Time postprandial (min) AUC (mL.min) [change from baseline] 0 60 120 180 Bread 47 (7) 24 (6) 4 (1) 13 (4) 32 (7) -5662 (1209) Lettuce 60 (14) 69 (13) 65 (14) 78 (16) 108 (13) 3194* (1574) Rhubarb 55 (9) 51 (8) 103 (11) 141 (16) 142 (12) 10586*# (1629)  P <0.01 versus bread; # P <0.01 vs. lettuce Table 1 – Fasting and postprandial Small Bowel Water Content (SBWC) in mL 23 Table 2 – T1 in milliseconds of Ascending Colon (AC) contents for the 3 test meals Mean (SEM) Fasting Time postprandial (min) AUC [change from baseline] 0 60 120 180 Bread 674 56) 726 (58) 771 (60) 750 (48) 785 (64) 15188 (6717) Lettuce 763 (65) 851 (75) 874 (54) 854 (61) 906 (83) 18990 (7884) Rhubarb 602 (73) 781 (71) 919 (56) 957 (70) 837 (66) 35744 (9013) able 2 – T1 in milliseconds of Ascending Colon (AC) contents for the 3 test Table 2 – T1 in milliseconds of Ascending Colon (AC) contents for the 3 test meals 24 Table 3 - AUCs and peak for ACWC, Gastric Volume, Bloating and Satiety Scores Table 3 - AUCs and peak for ACWC, Gastric Volume, Bloating and Satiety ACWC (mls) GV (mls) Bloating (VAS score) Satiety (VAS Score) Meal AUC Peak AUC Peak AUC Peak AUC Peak Bread 78 (43) 1 (T=0) 45195 (3297) 314 (T=0) 4196 (637) 27.6 (T=0) 7943 (783) 49.4 (T=0) Lettuce 409 (231) 5 (T=0) 64746 (4322) 570 (T=0) 7071 (905) 44.7 (T=0) 9498 (941) 64.8 (T=0) Rhubarb 291 (89) 3 (T=180) 67909 (3626) 558 (T=0) 5113 (927) 33.2 (T=0) 7904 (835) 47.5 (T=0) 25 25 Figures Figure 1: Consort Flow Diagram Figures Figure 1: Consort Flow Diagram Figures 26 Figure 2: Time course data for MRI measurements of small bowel water content (SBWC, meanSEM) when fasting and at intervals after isocaloric test meals of bread and butter, lettuce and mayonnaise or rhubarb and cream in 15 healthy adult subjects. 27 Figure 3: Time course data for MRI measurements of ascending colon water content (ACWC, meanSEM) when fasting and at intervals after isocaloric test meals of bread and butter, lettuce and mayonnaise or rhubarb and cream in 15 healthy adult subjects. 28 Figure 4: Time course data for T1 MRI longitudinal relaxation time of the chyme in the ascending colon (T1AC). Values (meanSEM) show change from fasting baseline after isocaloric test meals of bread and butter, lettuce and mayonnaise or rhubarb and cream in 15 healthy adult subjects.
https://openalex.org/W2593109333	https://upcommons.upc.edu/bitstream/2117/105382/1/sensors-17-00499.pdf	English	null	Analytical and Experimental Performance Evaluation of BLE Neighbor Discovery Process Including Non-Idealities of Real Chipsets	Sensors	2,017	cc-by	13,468	Analytical and Experimental Performance Evalu of BLE Neighbor Discovery Process Including Non-Idealities of Real Chipsets David Perez-Diaz de Cerio 1,, Ángela Hernández 2, Jose Luis Valenzuela 1 and Antonio Valdovinos 2 David Perez-Diaz de Cerio 1,, Ángela Hernández 2, Jose Luis Valenzuela 1 and Antonio Valdovinos 2 1 Signal Theory and Communications Department, Universitat Politècnica de Catalunya, C/Esteve Terrades 7 08860 Castelldefels, Spain; valens@tsc.upc.edu 2 Aragon Institute for Engineering Research (I3A), University of Zaragoza, 50018 Zaragoza, Spain; anhersol@unizar.es (Á.H.); toni@unizar.es (A.V.) * Correspondence: dperez@tsc.upc.edu; Tel.: +34-934-137-209 Academic Editor: Davide Brunelli Received: 23 December 2016; Accepted: 27 February 2017; Published: 3 March 2017 Abstract: The purpose of this paper is to evaluate from a real perspective the performance of Bluetooth Low Energy (BLE) as a technology that enables fast and reliable discovery of a large number of users/devices in a short period of time. The BLE standard speciﬁes a wide range of conﬁgurable parameter values that determine the discovery process and need to be set according to the particular application requirements. Many previous works have been addressed to investigate the discovery process through analytical and simulation models, according to the ideal speciﬁcation of the standard. However, measurements show that additional scanning gaps appear in the scanning process, which reduce the discovery capabilities. These gaps have been identiﬁed in all of the analyzed devices and respond to both regular patterns and variable events associated with the decoding process. We have demonstrated that these non-idealities, which are not taken into account in other studies, have a severe impact on the discovery process performance. Extensive performance evaluation for a varying number of devices and feasible parameter combinations has been done by comparing simulations and experimental measurements. This work also includes a simple mathematical model that closely matches both the standard implementation and the different chipset peculiarities for any possible parameter value speciﬁed in the standard and for any number of simultaneous advertising devices under scanner coverage. Keywords: Bluetooth Low Energy; neighbor discovery; non-detection probability; discovery latency; real testbed sensors sensors sensors 1. Introduction Bluetooth is a technology usually associated with short-range personal area networks with a low density of active devices, and many users only see it as the technology employed by their hands-free devices [1,2]. However, this vision of Bluetooth is changing with the appearance of the Internet of Things (IoT), connecting billions of devices and services anytime and everywhere, generating an incredible amount of new applications in many ﬁelds: wearable devices, health care, smart homes, connected cars, smart cities, etc. [3,4]. Some of the IoT applications [5], which require mobility and global connectivity based on a cellular network, will use 4G technologies such as LTE Machine to Machine (LTE-M) or Narrowband IoT (NB-IoT) and 5G technologies in the future. Nevertheless, most of the IoT devices will not use cellular wireless systems; instead, they will employ technologies that operate in Industrial Scientiﬁc Medical Sensors 2017, 17, 499; doi:10.3390/s17030499 www.mdpi.com/journal/sensors Sensors 2017, 17, 499 2 of 23 (ISM) unlicensed bands, such as Wi-Fi, ZigBee, Bluetooth and others, which will form what is known as capillary networks [6]. This is one of the reasons why Bluetooth introduced from its Version 4.0 [7] a low energy variant known as Bluetooth Smart or Bluetooth Low Energy (BLE). BLE includes lower power modes, lower complexity and lower cost, which are indispensable characteristics for making it a suitable technology for the IoT in the following years. gy g y As any emerging technology, BLE has raised a wide range of new questions and aspects that need to be characterized: its power efﬁciency, latency, range, throughput, etc. Searching for a global perspective that takes into account all of them for the whole variety of BLE-based services and applications is practically unapproachable. In [8], the BLE protocol stack is described, and the impact of several parameters on its performance is investigated, showing that conﬁguration parameters have a big impact on the tradeoff between performance and energy consumption. Many other studies evaluate new services and applications based on the BLE technology: healthcare, home automation, proximity sensing, consumer electronics, security, vehicular communications, sport, etc. Some examples are the BLE-based blood pressure monitoring system developed in [9], the remote lock system designed in [10] for pervasive computing environments, the fuzzy logic-based mechanism for energy management in home automation presented in [11] or the studies about the potential feasibility of BLE technology for intra-vehicular wireless sensor networks [12] and for inter-vehicular communications [13]. 1. Introduction Among that range of applications, we focus our analysis on high density scenarios. These involve the discovery of a large number of users/devices in a short period of time, such as race tracking for sport events, cattle control, access control, goods traceability, etc. The purpose of this paper is to evaluate the performance of BLE as a technology that enables fast and reliable discovery and identiﬁcation in high density environments from a real perspective, including the non-idealities present in off-the-shelf devices. The probability of not discovering a device in a given period of time depends on a vast number of variables and parameters. Some of them are technology dependent, such as the scan window, scan interval, advertising interval, frame size, advertising channels, transmitted power, etc., and others come determined by the environment: interference, noise, collisions, received signal level, etc. In this paper, we will analyze the probability of not detecting a frame of a system with multiple advertisers (devices transmitting beacon frames periodically) and one device continuously scanning for those frames. Once we obtain this probability, several other metrics will be derived, which result in being very useful for the IoT world, such as the average number of transmissions before detection, the detection latency, the average number of detections within a window of opportunity, etc. The importance of the topic and its expected impact in the following years is revealed by previous works in the literature, which focus on similar aspects, such as the modeling of the discovery latency. For example, [14] proposes an analytical model for evaluating a three-channel-based neighbor discovery procedure, further enhanced in [15] with real experiments to analyze the energy consumption. The work in [8] evaluates the impact of several of its parameters on its performance, including also real measurements of the energy consumption. Another example of the analysis of the latency performance combined with simulation experiments can be found in [16], where multiple BLE pairs (advertisers and scanners) are considered. The most recent work in [17] improves the models of [14,15] using the Chinese remainder theorem. This work analyzes the best tradeoff between discovery latency and energy consumption considering a variable scan window, scan interval and advertising intervals and concluding that the best conﬁguration comes when the advertising interval is equal to the scan window. 2. BLE Overview BLE was ﬁrst introduced in Version 4.0 of the Bluetooth Core Speciﬁcation [7]. The low energy system includes features designed to enable products that require lower current consumption, less complexity and reduced cost compared to Bluetooth legacy products. The system is also designed for use cases and applications with lower data rates and has shorter duty cycles. Like legacy Bluetooth, the BLE radio operates in the unlicensed 2.4 GHz ISM band using forty physical channels separated by 2 MHz. The neighbor discovery process is one of the main modiﬁcations that were introduced in BLE. Unlike classical Bluetooth, BLE dedicates three special channels (37, 38 and 39), called advertising channels, to the discovery of other BLE devices, initiating a connection and broadcasting data. Channels from 0 to 36 are used for data exchange between connected devices. The BLE system employs a frequency hopping transceiver to combat interference and fading, but this mechanism is not employed on the advertisement transmissions. Hence, the advertising channels have been strategically chosen to reduce interference from other systems that coexist in the 2.4 GHz band. Low energy radio operation uses a Gaussian Frequency Shift Keying (GFSK) modulation to minimize transceiver complexity. The symbol rate is 1 Mbaud and supports a bitrate of 1 Mbps. The objective of this work is to discover and identify the devices present in the operating scenario in the shortest possible time, but with no additional data exchange. Due to this, for the purpose of the paper, only non-connectable undirected advertising events are relevant, as no connections will be established in any case. Devices that transmit advertising packets on the advertising PHY channels are referred to as advertisers, whereas the devices that enter in scanning mode, wanting to discover its neighbors without the intention of establishing a connection after the discovery, are referred to as scanners. As shown in Figure 1, in order to announce its presence, the advertiser periodically generates non-connectable undirected advertising events, which consist of a sequence of packets in the three advertising channels indexed by 37, 38 and 39. That is, the PHY channel changes for each packet sent by the advertiser in the same event following a round-robin cycle 37 →38 →39 →37 → 38 →39 →· · · . The successive advertisement events are separated by a predeﬁned advertisement interval. 1. Introduction All of the aforementioned works are based on the premise that the scanner behavior matches exactly what is derived from the standard and do not consider non-idealities present in real devices. For example, one could consider that when the scan window is matched with the scan interval, the scanner should be doing a continuous scan, i.e., scanning 100% of the time. However, along the paper, we will demonstrate that this is not true, and the impact of these non-idealities is very signiﬁcant on the results and calculations derived from them. Measurements show that additional gaps appear in 3 of 23 Sensors 2017, 17, 499 the scanning process, which reduce the discovery capabilities. These gaps have been identiﬁed in all of the measured devices and respond to both regular patterns and variable events associated with the decoding process. In addition, we propose a simple mathematical model for the non-detection probability and the main performance parameters derived from it, which includes both the standard implementation and the non-idealities present in the devices. The results have been validated with extensive experimentation in a real testbed, and simulations have been carried out to extrapolate the results for high density scenarios. The mathematical model will be a valuable tool to easily obtain an upper bound of discovery capacities and to select the desirable parameters values according to a particular BLE application. The structure of the paper is as follows. In Section 2, we shortly describe the standard BLE, and we present the experimental analysis performed in order to characterize the non-idealities existing in the manufactured chipset scanner operation. In Section 3, we propose a simple mathematical model that meets both the standard implementation and the manufactured chipset peculiarities. Numerical results from the mathematical model, the simulation and the experimental measurements of real devices are presented and discussed in Section 4. Finally, the main conclusions are summarized in Section 5. 2. BLE Overview To avoid repeated collisions between simultaneous advertising devices, a random delay is added to the advertisement interval. The scanner enters into the scanning mode in periodic time intervals called the scan interval and listens to the advertising messages for a ﬁxed time called the scan window. The scanner changes its scanning channel after each scan interval following the already explained sequence. p q Depending on the type of advertising packet, the scanner may make a request to the advertiser on the same advertising PHY channel, which may be followed by a response from the advertiser. In this study, we analyze only non-connectable advertising events. 4 of 23 Sensors 2017, 17, 499 Advertising event Advertising event Advertising event advInterval advInterval T_advEvent T_advEvent advDelay advDelay ADV_IND PDU ADV_IND PDU ADV_IND PDU ≤ 10 ms ≤ 10 ms Channel 37 Channel 38 Channel 39 tadv Preamble 1 Byte Access address 4 Bytes Packet Data Unit (PDU) Advertising data 8 ‐39 Bytes CRC 3 Byte Header 2 Bytes ADV address 6 Bytes ADV struct Flags 3 Bytes ADV Struct specific data Length 1 Byte Type 1 Byte Data 1 Byte Length 1 Byte Type 1 Byte Data 1‐ 26 Bytes Advertiser Scanner Scan window Scan interval Scan window Scan window Channel 38 Channel 37 Channel 39 Scan interval Figure 1. Transmissions on BLE advertising PHY channels and the frame structure. Scanner Advertiser ADV_IND PDU ADV_IND PDU Access address 4 Bytes ADV struct Flags 3 Bytes Figure 1. Transmissions on BLE advertising PHY channels and the frame structure. The advertising frame or advertising Packet Data Unit (PDU) follows the structure depicted in the lowest part of Figure 1. The payload is variable from eight to 39 raw bytes; however, in our experiments, the payload consists of the ADV address, a three-byte ﬂags ﬁeld to allow the reading of the advertising by standard mobile applications and the data itself. Since the transmission bitrate is 1 Mbps, Table 1 summarizes the duration of the frames for the three data sizes employed in our analysis. Table 1. Frame duration for the employed sizes. Table 1. Frame duration for the employed sizes. Data Bytes Total Frame Bytes Duration 1 22 176 µs 10 31 248 µs 26 47 376 µs 2.1. Characterization of Non-Idealities Present in Real Devices 2.1. Characterization of Non-Idealities Present in Real Devices 2.1. Characterization of Non-Idealities Present in Real Devices 2.1. Characterization of Non-Idealities Present in Real Devices From our previous experience, we know that the behavior of real devices is not ideal. For example, one could suppose that setting the same value to the scan window (TscanWindow) and scan interval (TscanInterval) in the scanning device would keep the receiver in a continuous scan mode. However, this is not true, and the devices present short intervals during which they are not scanning. Some of these gaps are present even when there is only the scanner in the scenario, while others appear when the scanner decodes advertising frames. These gaps should not be ignored because even if part of a frame is received during a gap, the frame would not be detected. Thus, below, we will proceed to the characterization of these gaps, differentiating the pauses exclusively due to the receiver and the ones appearing because of processing frames. To carry out this characterization, we analyzed the instantaneous power consumption of the devices using a current sensing solution based on the design of [18], which accurately detects load currents between 0 A and 1 A. Unlike the work in [15], in our case, the aim is not to analyze the power consumption of the devices, but instead, to extract behavior 5 of 23 Sensors 2017, 17, 499 patterns that allow us to determine the amount of time during which a frame cannot be detected. The ﬁnal implementation of the sensor testbed is shown in Figure 2. Current sensors RF Shield box BLE scanner BLE advertisers Power supply Digital oscilloscope Laptop Figure 2. Testbed schematic setup. Figure 2. Testbed schematic setup. 2.1.1. Scanning Gaps Caused Exclusively by the Scanning Device We analyzed the performance of several devices of different BLE chipset manufacturers, including Broadcom, Cambridge Silicon Radio, Nordic Semiconductor, Bluegiga and Texas Instruments. We conﬁgured all of them to scan continuously, i.e., ﬁxing the scan window and the scan interval to the same value. The reason behind this decision is to avoid other effects that would hinder the desired characterization, such as the ﬂuctuation phenomenon described in [17,19] under speciﬁc combinations of these two values. Once the devices are characterized, analyzing the behavior for the unmatched scan window and scan interval values can be easily performed. From the observed measurements, we have concluded that all scanning devices follow with slight variations two behavior patterns, referred to as Type 1 and Type 2. The ﬁrst pattern schematic is shown in Figure 3a, where it can be observed that a unique gap appears in every scan interval. This pause is associated with the change of the scanning frequency. It has been measured that for all Type 1 devices, this gap has a ﬁxed duration of Tf qChgGap = 1.1 ms, regardless of the employed scan interval. TfqChgGap TfqChgGap TscanWindow=TscanInterval (a) TinterFqChgGap TgapInt1 TgapInt1 TgapInt2 TfqChgGap TfqChgGap TscanWindow=TscanInterval (b) Figure 3. Continuous scan behavior. (a) Type 1 scanning devices; (b) Type 2 scanning devices. (b) Figure 3. Continuous scan behavior. (a) Type 1 scanning devices; (b) Type 2 scanning devices. 6 of 23 Sensors 2017, 17, 499 On the other hand, other devices follow the pattern shown in Figure 3b. As can be observed, the scanning frequency change gap is also present, but there are also other periodic pauses of less duration, which also affect the performance of the system. We will name TinterFqChgGap the duration of these gaps and TgapInt1 and TgapInt2 the periods between them. It must be noted that the pattern shows a peculiar event (highlighted in red) where only one gap of duration TinterFqChgGap is present when two of them separated by TgapInt2 were expected. This effect happens always in the penultimate group of double gaps. The authors want to remark that the description of the device behavior presented here is based on real measurements. This behavior depends on the speciﬁc device ﬁrmware and hardware, which is designed and implemented by the chipset manufacturers. 2.1.1. Scanning Gaps Caused Exclusively by the Scanning Device As an example, the numerical values for the reference device, which follows the Type 2 pattern for a TscanWindow = TscanInterval = 500 ms, are shown in Table 2. However, as opposed to the devices that follow the Type 1 pattern, Tf qChgGap depends on the scan interval used, as depicted in Figure 4. Table 2. Parameter values for a continuous scan with TscanWindow = TscanInterval = 500 ms. Table 2. Parameter values for a continuous scan with TscanWindow = TscanInterval = 500 ms. Parameter Value Type 1 Pattern Type 2 Pattern Tf qChgGap 1.1 ms 16.05 ms TinterFqChgGap - 300 µs TgapInt1 - 16.82 ms TgapInt2 - 4.3 ms Figure 4 depicts the evolution of Tf qChgGap versus the selected scan interval. As stated before, the Tf qChgGap duration for the devices that follow the Type 1 pattern is constant, and the ones following the Type 2 pattern increase linearly. In the same ﬁgure, the percentage between Tf qChgGap and the scan interval is shown, reﬂecting the importance of these gaps especially for low scan interval values. 0 20 40 60 80 100 120 140 0 0.6 1.2 1.8 2.4 3 3.6 4.2 0 500 1000 1500 2000 2500 3000 3500 4000 "Type 1 pattern" gap size (TfqChgGap) "Type 2 pattern" gap size (TfqChgGap) "Type 1 pattern" percentual gap size "Type 2 pattern" percentual gap size Gap size (ms) Percentual size Scan window and scan interval (ms) Figure 4. Frequency change gap duration for different scan intervals. Percentual size Figure 4. Frequency change gap duration for different scan intervals. Figure 4. Frequency change gap duration for different scan intervals. p y • The advertisement frame length does not have an effect in any case 2.1.2. Scanning Gaps Caused by the Processing of Frames So far, it may seem that devices following the Type 1 pattern have better performance than devices following the second pattern; however, next, we will see that global performance depends also on the 7 of 23 Sensors 2017, 17, 499 number of received frames. This happens because when a frame is received, the scanner abandons momentarily the scanning state to process the frame; producing, in this way, different pauses from the already analyzed ones. To characterize this behavior, we followed the schema depicted in Figure 2. Now, we included 14 devices manufactured by RedBearLab, speciﬁcally the BLE Nano based on Nordic nRF51822 SoC [20] as advertisers and a second current sensor for measuring simultaneously in the oscilloscope the behavior of the advertisers and the scanner. In this setup, the laptop only controlled and powered the scanner. Figure 5 is an example where the consumption of an advertiser in channels 37, 38 and 39 (green line) and the behavior of a scanner based on the Type 1 pattern (blue line) are depicted. In red, the start and the end of the gap due to the frame processing are represented. As can be observed, at that moment, the receiver was scanning Frequency 38, as it detected the second frame of the advertisement event. These gaps are also dependent on the device. In order to determine their statistics, we implemented an automatized measurement process to analyze the duration of the pauses for different frame sizes and for the two reference devices (Type 1 and Type 2 patterns). To calculate the duration and to discard invalid samples, we apply a step detector in the transmitted and received signal. We consider the beginning of the gap when a falling edge of the transmitted signal coincides with another falling edge of the received signal within a margin of 200 samples (20 µs). The end of the gap is determined by the immediately next rising edge of the received signal. The realizations were formed by 1000 samples, and the step detector implemented was based on the Canny algorithm for one dimension [21]. 0 0.05 0.1 0.15 0.2 0.25 0 50 100 150 200 250 300 350 400 450 500 RX- type 1 scanning device TX- advertising device Time (ms) Voltage (V) 0.958ms Figure 5. Consumption example of the advertiser/scanner. Figure 5. Consumption example of the advertiser/scanner. 2.1.2. Scanning Gaps Caused by the Processing of Frames From this analysis, we can extract the following conclusions depicted in Figure 6: From this analysis, we can extract the following conclusions depicted in Figure 6: • The reference device with the Type 1 pattern has a uniform distributed processing gap duration (τdecodGap) with a minimum value (TminDecodGap) of 350 µs and a maximum value (TmaxDecodGap) of 1.6 ms. • For the second device (Type 2 pattern), the duration of the pause is considerably lower (194 µs) and is practically constant. p y • The advertisement frame length does not have an effect in any case. 8 of 23 Sensors 2017, 17, 499 0 1 2 3 4 5 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 Type 1 scanning device Frequency of gap size (%) 2 Processing gap size (ms) (a) 0 20 40 60 80 100 0 0.04 0.08 0.12 0.16 0.2 0.24 0.28 0.32 0.36 Type 2 scanning device Frequency of gap size (%) Processing gap size (ms) 0.4 (b) Figure 6. Continuous scan behavior. (a) Type 1 pattern; (b) Type 2 pattern. 0 1 2 3 4 5 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 Type 1 scanning device Frequency of gap size (%) 2 Processing gap size (ms) (a) 0 20 40 60 80 100 0 0.04 0.08 0.12 0.16 0.2 0.24 0.28 0.32 0.36 Type 2 scanning device Frequency of gap size (%) Processing gap size (ms) 0.4 (b) (b) (a) Figure 6. Continuous scan behavior. (a) Type 1 pattern; (b) Type 2 pattern. Figure 7 summarizes the combined effect of both gap types for the ﬁrst reference device in a scenario with several advertisers (blue line). A total of ten interruptions of the scanning (yellow line) can be observed. We will analyze each of these pauses with the corresponding advertisement events prior to the gap. 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th Figure 7. Combined effect of both types of gaps. Figure 7. Combined effect of both types of gaps. All of the pauses correspond to processing gaps except from the penultimate (i.e., the ninth gap), which is due to the change of scanning frequency. 2.1.2. Scanning Gaps Caused by the Processing of Frames At the beginning of the capture, it can be observed that the receiver is scanning at Frequency 39 because the processing frame gap is present in the last frame of the advertisement events, and after it (last gap), the detected frame is the ﬁrst of the advertisement event. If we analyze the rest of the gaps in detail, we can see that the ﬁrst gap comes after an overlap of two advertisements events. From these two events, only the ﬁrst is detected, while the second one Sensors 2017, 17, 499 9 of 23 is missed because the last frame of the event, which matches the current scanning frequency, arrives while the receiver is still processing the frame of the ﬁrst event. is missed because the last frame of the event, which matches the current scanning frequency, arrives while the receiver is still processing the frame of the ﬁrst event. The second, third, fourth, sixth and eighth gaps correspond to the detection of single advertisements events on Frequency 39. It should be noticed that their duration is variable. The ﬁfth and seventh gaps also correspond to an overlap of several advertisement events, like the ﬁrst. However, in the last ones, the number of advertisement events overlapping is greater attending to the amplitude depicted. In both cases, only one of the advertisements is detected, while the rest are not because the receiver is processing the detected frame. Therefore, although the ﬁrst impression is that the scanners that follow the Type 1 pattern have better performance because their scanning frequency change gaps are smaller, in scenarios with a high density of transmitters, the processing gaps gain importance, and both effects should be considered. 3. Analytical Model Variables included in the mathematical model. Variable Description Ppattern scanGap Probability that a periodical scanning gap occurs PdecodGap Probability that a device causes a scanning interruption between two consecutive advertisements NNBLE decodGap Average number of devices that may be generating decoding gaps within a TadvInterval + τadvDelay interval in a scenario with NBLE advertisers PscanGap noDetect Non-detection probability due to periodical scanning gaps PdecodGap,NBLE noDetect Non-detection probability due to dynamic scanning gaps (decoding gaps) in an scenario with NBLE advertisers Pgap,NBLE noDetect Non-detection probability due to scanning gaps (periodical and dynamic scanning gaps) in a scenario with NBLE advertisers Pcol,NBLE noDetect Non-detection probability of a device due to collisions in a scenario with NBLE advertisers Pgap+col,NBLE noDetect Non-detection probability of a device due to collisions and gaps in a scenario with NBLE advertisers PNBLE noDetect Overall non-detection probability of a device due to collisions, gaps and channel errors in a scenario with NBLE advertisers NNBLE advReq Average number of ADV_PDU transmissions required before detection of a device in a scenario with NBLE advertisers DNBLE detect Average detection delay of a device in a scenario with NBLE advertisers tNBLE interDetect Average time between two consecutive detections of a device in a scenario with NBLE advertisers NNBLE detect Average number of detections of an advertiser BLE within a window of opportunity (coverage time interval or dwell time) in a scenario with NBLE advertisers The number of BLE devices involved in the analysis is NBLE + 1. A ﬁxed device is conﬁgured as a scanner, whereas the other NBLE devices are conﬁgured as advertisers. The number of BLE devices involved in the analysis is NBLE + 1. A ﬁxed device is conﬁgured as a scanner, whereas the other NBLE devices are conﬁgured as advertisers. In the ﬁrst approach and in order to meet the requirements of applications where a number of users need to be counted in a very short period of time, the scanner BLE ﬁxes the same scan window and scan interval values to keep the receiver scanning 100% of the time. A collision occurs when the ADV_PDU transmissions of at least two advertisers are time overlapped on the same channel that the receiver is scanning. We assume that the three channels are equally interfered and that all of the devices keep the same event advertising timings. 3. Analytical Model In this section, our objective is to deﬁne a simple, but still accurate mathematical model to describe the BLE device discovering process in full accordance with the BLE speciﬁcation and be able to reﬂect the behavior particularities of different BLE chipset manufacturers. We start from a simple long-term characterization of the collision probability according to the ideal implementation of the BLE speciﬁcation. Then, we use it as a basis to derive the non-detection probability by including the required adaptations to meet the non-idealities of real BLE chipset manufacturer implementations presented in Section 2. Note that the non-detection probability depends on three main components: collisions between advertisements packets, gaps in the scanning process of the scanner BLE and Block Error Rate (BLER) due to channel and interference conditions. Next, we derive several parameters of interest, such as the mean discovery latency and mean number of detections in the time spent (dwell time) of an advertising BLE under the scanner coverage area. Finally, we prove that the proposed model agrees with experimental measurements and the results derived from simulations for a small and a high number of competing BLE devices. The analytical model used for non-detection probability characterization takes into account the parameters and variables summarized in Tables 3 and 4. Table 3. Parameters included in the model. Parameter Description Tadv Transmission time of the advertisement PDU (ADV_PDU) TadvInterval Fixed advertisement interval TadvDelayMax Maximum value of the random backoff (standard: 10 ms) τadvDelay Random backoff between advertisements: Uni f orm[0, TadvDelayMax] TadvEvent Advertisement event interval: TadvInterval + τadvDelay TscanInterval Scan interval TscanWindow Scan window Tf qChgGap Gap due to change of scanning frequency (Type 1 and 2 scanners) TinterFqChgGap Duration of scattered gaps inside the scan interval (Type 2 scanner) TgapInt1, TgapInt2 Time intervals between scattered gaps inside the scan interval (Type 2 scanner) TminDecodGap Minimum value of processing gap after ADV_PDU detection TmaxDecodGap Maximum value of processing gap after ADV_PDU detection τdecodGap Processing gap after ADV_PDU detection. Uni f orm[TminDecodGap, TmaxDecodGap] TscanGap Sum of durations of all the gaps occurred in the scan window NscanWindow interFqChgGap Number of scattered gaps inside the TscanWindow NBLE Total number of advertising devices that are in the coverage area of the scanner and can be potentially colliding Table 3. Parameters included in the model. Table 3. Parameters included in the model. 10 of 23 10 of 23 Sensors 2017, 17, 499 Table 4. 3. Analytical Model Then, we characterize the collision probability as if the scanner were always scanning in the same channel and the advertisers were always transmitting in this frequency. In order to develop the analytical model, we note that in both the ideal and the real implementation, the advertisement process for a device is independent of other devices, and it is not conditioned by collisions or non-detections. At a determined time instant, the collision probability of the ADV_PDU transmission of a device with the ADV_PDU of other advertiser BLE devices is a stochastic process with memory. The advertisement event interval (TadvEvent) is the sum of a ﬁxed component (TadvInterval) and a pseudo random variable (τadvDelay), and the transmission time of the ADV_PDU is ﬁxed. Thus, the process differs from a Markovian process. The collision probability of an ADV_PDU depends on the history of transmissions of all BLE devices that coexist under the scanner coverage area. For instance, in a straightforward scenario where NBLE = 2 and the two BLE devices enter into coverage of the BLE scanner, the time difference among their ﬁrst advertisements is conﬁned to the interval [0, TadvInterval + TadvDelayMax]. However, if the two BLE devices have previously collided in the ﬁrst 11 of 23 Sensors 2017, 17, 499 transmission (1st_Adv_Tx), the time difference among their respective sequent advertisement events (2nd_Adv_Tx) is a random variable conﬁned to the time interval [0, TadvDelayMax]. In the same way, the time difference interval between the advertisements of the two devices in a 3rd_Adv_Tx, after a collided transmission in the 1st_Adv_Tx and a non-collided transmission in the 2nd_Adv_Tx, is conﬁned to a time interval up to [0, 2 · TadvDelayMax −Tadv], and so on. y However, in a long-term and averaged analysis, we can simplify the collision probability calculation. When a collision occurs, the probability that the transmission of a reference advertiser (started in a time instant t) collides with another one, Pcol,2 noDetect, corresponds with the probability that one neighbor device starts its own transmission in the interval [t −Tadv, t + Tadv]. As the mean time between consecutive advertisement transmissions is TadvInterval + τadvDelay, the probability of collision is 2·Tadv TadvInterval+τadvDelay . Finally, given that transmissions of the NBLE devices are independent, the collision probability of a reference advertiser with any other of its neighbor devices is one minus the probability of not colliding with any of them. 3.1. Non-Detection Probability According to the Behavior of the Actual Chipsets Now, we will include in the non-detection probability characterization the behavior particularities of different BLE chipset manufacturers. As we have seen in Section 2, the scanners present short pauses on the scanning, which we have compiled into two possible behavior patterns, shown in Figure 3. The effects of these gaps on the non-detection probability will be included in the model by treating separately the existence of the two types of pauses in which the scanner does not scan. That is: The periodical scanning gaps (with ﬁxed duration): which correspond to the ﬁxed periods of time in which the scanner does not scan. They are present even when there is only the scanning device in the scenario. The non-detection probability due to these periods will be denoted by PscanGap noDetect. the scenario. The non-detection probability due to these periods will be denoted by PscanGap noDetect. The dynamic scanning gaps (with ﬁxed or variable duration): These interruptions in the scanning process occur every time the scanner correctly detects a frame. As a consequence, they depend on the number of advertisements the scanner is detecting and thus on the number of simultaneous BLE advertisers under the scanner coverage. The non-detection probability due to these periods in a scenario with NBLE advertising devices will be denoted by PdecodGap,BLE noDetect . In the next sections, we describe all considerations that allow us to derive the ﬁnal expression of PscanGap noDetect and PdecodGap,BLE noDetect . Ppattern scanGap = TscanGap TscanWindow = Tf qChgGap+NscanWindow interFqChgGap·TinterFqChgGap TscanWindow (3) 3. Analytical Model Thus, the collision probability in a scenario with NBLE advertisers is obtained with Equation (1). Pcol,NBLE noDetect = 1 −(1 −Pcol,2 noDetect)NBLE−1 with Pcol,2 noDetect = 2·Tadv TadvEvent = 2·Tadv TadvInterval+τadvDelay (1) (1) Next, the characterization of interference from other communication systems and path loss effects can be included by means of a BLER parameter on the unsuccessful reception probability of an ADV_PDU. Non-detection probability in a scenario with NBLE advertisers (PNBLE noDetect), including collision effects and errors when ADV_PDUs do not collide, is obtained by Equation (2): Next, the characterization of interference from other communication systems and path loss effects can be included by means of a BLER parameter on the unsuccessful reception probability of an ADV_PDU. Non-detection probability in a scenario with NBLE advertisers (PNBLE noDetect), including collision effects and errors when ADV_PDUs do not collide, is obtained by Equation (2): PNBLE noDetect = Pcol,NBLE noDetect + (1 −Pcol,NBLE noDetect ) · BLER (2) (2) 3.1. Non-Detection Probability According to the Behavior of the Actual Chipsets 3.1.1. Periodical Scanning Gaps In a ﬁrst approximation, assuming that the scanner is alone, the probability of a gap of this type occurring (Ppattern scanGap) is the ratio between the sum of the mean durations of all of the gaps occurring on In a ﬁrst approximation, assuming that the scanner is alone, the probability of a gap of this type occurring (Ppattern scanGap) is the ratio between the sum of the mean durations of all of the gaps occurring on the scan window (denoted as TscanGap) and the TscanWindow. Ppattern scanGap is calculated with Equation (3), being NscanWindow interFqChgGap the number of scattered gaps inside the TscanWindow and conveniently derived from using the T T T T and T parameters the scan window (denoted as TscanGap) and the TscanWindow. Ppattern scanGap is calculated with Equation (3), being NscanWindow interFqChgGap the number of scattered gaps inside the TscanWindow and conveniently derived from using the TscanWindow, Tf qChgGap, TinterFqChgGap, TgapInt1 and TgapInt2 parameters. (3) 12 of 23 Sensors 2017, 17, 499 However, starting here, the question is what happens when simultaneous advertiser devices are present in the system and how these gaps on the scanning affect the detection. In this case, we will see that the operation mode of the scanner simpliﬁes the characterization of this impact. We have veriﬁed that if an advertisement is sent and during the reception time, the scanner has planned a periodic gap, two possible overlapping cases can occur, with different effects: 1. If ADV_PDU transmission starts later than the start time of a periodic gap, the ADV_PDU is not detected. In this case, the time intervals between consecutive gaps (TgapInt1 and TgapInt2) are not affected. 1. If ADV_PDU transmission starts later than the start time of a periodic gap, the ADV_PDU is not detected. In this case, the time intervals between consecutive gaps (TgapInt1 and TgapInt2) are not affected. 2. If ADV_PDU frame reception is earlier than the start time of a periodic gap, this gap is postponed until the end of reception, regardless of whether the reception is correct, or an error, or a collision has occurred. In this case, the time interval between the postponed periodic gap and the following gap is reduced with respect TgapInt1 or TgapInt2, because the planned start times of the following gaps remain unchanged according to the pattern timing deﬁned in the scanner device. 3.1.1. Periodical Scanning Gaps Moreover, we know that after successful advertisement receptions, the scanner introduces a gap related to the decoding time. In this case, when the reception is correct, the decoding gap and also the postponed gap scan should start simultaneously, but the scanner only applies the larger of them. On the other hand, if an unsuccessful reception or a collision occurs, the periodic scan gap is applied after the last frame involved in the collision is received. The behavior described here is based on the analysis of real measurements of a testbed involving up to 14 simultaneous devices operating under the parameters of the standard. This behavior depends on the ﬁnal manufacturer implementation of the ﬁrmware and hardware. tt Furthermore, Ppattern scanGap remains unchanged, according to (3). Therefore, the non-detection probability (PscanGap noDetect), according to Equation (4), depends on the probability of generating an advertisement within the scanning gap period and is the same as Equation (3). Note that according to the characterizations made in Section 2, Tf qChgGap and TinterFqChgGap are usually constant. PscanGap noDetect = TscanGap TscanWindow = Tf qChgGap+NscanWindow interFqChgGap·TinterFqChgGap TscanWindow (4) (4) 3.1.2. Dynamic Scanning Gaps These interruptions in the scanning process occur when the receiving device correctly detects a frame. In order to derive PdecodGap,NBLE noDetect , we ﬁrst obtain the probability that a device causes a scanning interruption between two consecutive advertisements (PdecodGap). In the ﬁrst approach, if the ADV_PDUs are always correctly detected, this corresponds to Expression (5): PdecodGap = τdecodGap TadvInterval+τadvDelay (5) (5) Nevertheless, since the advertisement transmission of a device has a certain probability of not being detected in the presence of NBLE devices (including itself), the expression to be handled in order to characterize the probability that the scanner enters in a decoding gap due to this advertiser is obtained by Equation (6): PdecodGap = τdecodGap TadvInterval+τadvDelay = τdecodGap·(1−PNBLE noDetect) TadvInterval+τadvDelay (6) (6) Starting from PdecodGap, we obtain the probability that the scanner enters in a decoding gap in a scenario with NBLE advertisers. In this case, two advertisers cannot simultaneously generate a decoding gap, but in the period of time between two advertisements of a reference device, its neighbor devices may trigger NBLE −1, NBLE −2, · · · , 1 or no decoding gaps on the scanner, depending on their detection probability. Therefore, we can calculate the mean time that the scanner generates a decoding gap within a TadvInterval + τadvDelay interval by multiplying the average time of the decoding gap after 13 of 23 Sensors 2017, 17, 499 ADV_PDU detection (τdecodGap) by the average number of devices that may be generating decoding gaps (NNBLE decodGap), according to Equation (7). Note that the system includes a ﬁnite population of advertising devices, so the probability of having n decoding gaps is a binomial distribution. Finally, the non-detection probability due to decoding gaps (PdecodGap,NBLE noDetect ) is the probability of generating an advertisement within a decoding gap period, which in this case is the probability of the scanner being in a decoding gap period, given by Equation (8). ADV_PDU detection (τdecodGap) by the average number of devices that may be generating decoding gaps (NNBLE decodGap), according to Equation (7). Note that the system includes a ﬁnite population of advertising devices, so the probability of having n decoding gaps is a binomial distribution. 3.1.2. Dynamic Scanning Gaps Finally, the non-detection probability due to decoding gaps (PdecodGap,NBLE noDetect ) is the probability of generating an advertisement within a decoding gap period, which in this case is the probability of the scanner being in a decoding gap period, given by Equation (8). NNBLE decodGap = NBLE−1 ∑ n=1 n · NBLE −1 n ! · (1 −PNBLE noDetect)n · PNBLE noDetect (NBLE−1)−n (7) (7) PdecodGap,NBLE noDetect = τdecodGap·NNBLE decodGap TadvInterval+τadvDelay (8) (8) It is important to keep in mind that PdecodGap,NBLE noDetect depends on PNBLE noDetect, and at the same time, PdecodGap,NBLE noDetect , in addition to PscanGap noDetect, will modify the probability PdecodGap,NBLE noDetect . Therefore, a recursive process will be used to obtain them. d d Once PscanGap noDetect and PdecodGap,NBLE noDetect are obtained, we must take into account the probability that the scanner is in a scanning gap, regardless of whether it is a decoding gap or in a gap due to its scan interruption pattern. According to the analysis made above, the two effects are characterized as independent, by they could occur simultaneously. Thus, the approach (9) is used in order to compute the non-detection probability (Pgap,NBLE noDetect ) due to both effects. Pgap,NBLE noDetect = PscanGap noDetect + PdecodGap,NBLE noDetect −PscanGap noDetect · PdecodGap,NBLE noDetect (9) (9) Then, the probability of non-detection due to collisions and gaps (Pgap+col,NBLE noDetect ) can be obtained with Equation (2), by setting Pgap,NBLE noDetect instead of BLER. Pgap+col,NBLE noDetect = Pcol,NBLE noDetect + (1 −Pcol,NBLE noDetect ) · Pgap,NBLE noDetect (10) (10) Finally, we will use the equations above to obtain PNBLE noDetect in an iterative way according to the algorithm shown in (11). 3.1.2. Dynamic Scanning Gaps n ←0 Pgap+col,NBLE noDetect (n) ←Pcol,NBLE noDetect (n) + 1 −Pcol,NBLE noDetect (n) · PscanGap noDetect(n) n ←n + 1 n ←0 Pgap+col,NBLE noDetect (n) ←Pcol,NBLE noDetect (n) + 1 −Pcol,NBLE noDetect (n) · PscanGap noDetect(n) n ←n + 1 n ←0 Pgap+col,NBLE noDetect (n) ←Pcol,NBLE noDetect (n) + 1 −Pcol,NBLE noDetect (n) · PscanGap noDetect(n) 1 n ←n + 1 obtain PdecodGap,NBLE noDetect (n) with Equations (7) and (8) Pgap,NBLE noDetect (n) ←PscanGap noDetect(n) + PdecodGap,NBLE noDetect (n) −PscanGap noDetect(n) · PdecodGap,NBLE noDetect (n) Pgap+col,NBLE noDetect (n) ←Pgap+col,NBLE noDetect (n −1) + 1 −Pgap+col,NBLE noDetect (n −1) · Pgap,NBLE noDetect (n) while Pgap+col,NBLE noDetect (n) −Pgap+col,NBLE noDetect (n −1) > ε (11) (11) n ←n + 1 obtain PdecodGap,NBLE noDetect (n) with Equations (7) and (8) Pgap,NBLE noDetect (n) ←PscanGap noDetect(n) + PdecodGap,NBLE noDetect (n) −PscanGap noDetect(n) · PdecodGap,NBLE noDetect (n) Pgap+col,NBLE noDetect (n) ←Pgap+col,NBLE noDetect (n −1) + 1 −Pgap+col,NBLE noDetect (n −1) · Pgap,NBLE noDetect (n) } } Pgap+col,NBLE noDetect ←Pgap+col,NBLE noDetect (n) } Pgap+col,NBLE noDetect ←Pgap+col,NBLE noDetect (n) S 2017 17 499 14 f 23 Sensors 2017, 17, 499 14 of 23 Similar to Equation (2), characterization of interference from other communication systems and path loss effects can also be included as the input BLER parameter in order to compute the overall non-detection probability using (12): PNBLE noDetect = Pgap+col,NBLE noDetect + (1 −Pgap+col,NBLE noDetect ) · BLER (12) (12) 3.2. Derived Parameters of Interest 3.2. Derived Parameters of Interest In this section, we derive several parameters of interest, such as the mean discovery latency and the mean number of detections during the time that an advertising BLE is under the scanner coverage area (dwell time), when there are NBLE simultaneous advertisers. N The average number of ADV_PDU transmissions required before detection (NNBLE advReq) can be straightforwardly obtained by: NNBLE advReq = 1 · 1 −PNBLE noDetect + ∞ ∑ k=1 (1 + k) · 1 −PNBLE noDetect PNBLE noDetect k = 1 + PNBLE noDetect 1−PNBLE noDetect (13) (13) The average detection delay (DNBLE detect) is deﬁned as the interval between when the advertiser transmits the ﬁrst advertising packet until the scanner receives its ADV_PDU correctly. Using Equation (13): DNBLE detect = NNBLE advReq −1 · TadvEvent (14) (14) om (13), the average time between two consecutive detections (TNBLE interDetect) is given by on (15): From (13), the average time between two consecutive detections (TNBLE interDetect) is given by Equation (15): TNBLE interDetect = NNBLE advReq · TadvEvent (15) (15) Finally, if we deﬁne a window of opportunity (coverage time interval or dwell time), TcovWindow, the average number of detections (NNBLE detect) of an advertiser BLE will be: NNBLE detect = TcovWindow TNBLE interDetect (16) (16) 4. Experimental and Simulation Validation of the Analytical Model The device discovery process for BLE as described in Section 2 is fairly simple, and in fact, this is the reason why we want to explore potential beneﬁts of using this process in applications where a certain number of users need to be counted in a very short period of time, as the sport applications referred to in Section 1. According to the requirements of these application scenarios, we want to quantify the discovery process for a large number of devices. The main parameters of the performance evaluation include: The collision probability/non-detection probability. • The collision probability/non-detection probability. The average discovery latency or, alternatively, the mean time between consecutive ADV_PDU detections of an advertiser. • The mean number of ADV_PDU detections within a window of opportunity time (coverage time interval or dwell time). Additional parameters, such as the probability that all coexisting advertisers can be detected by the scanner within the coverage time, are also included. Even in ideal conditions, with respect to wireless channel effects or interference from other systems, the performance of the device discovery process is greatly inﬂuenced by the scanner parameter settings, such as the scanning interval, the 15 of 23 15 of 23 Sensors 2017, 17, 499 scan window size and, in the advertisers, the advertising interval and the advertising PDU size. Speciﬁcally, concerning the scanning device, by choosing the scanning interval and the scan window size, we can control the tradeoff between the discovery capability and the energy consumption of the scanner. That is, for a given scanning interval, shortening the scan window, the energy consumption of the scanner is decreased, whereas the non-detection probability of the advertising devices increases. Nevertheless, from the point of view of these speciﬁc applications, the energy consumption is not one of the most relevant parameters, given that the main objective is to detect a high number of BLE devices in a short time window, even if we detect the identiﬁer of the same device several times. Repetition will provide us a desirable redundancy in order to compensate even high BLER scenarios that extend the experimental conditions and simulation analysis performed here. Thus, as explained in Section 2.1.1, to avoid other effects that would make the desired characterization difﬁcult, we ﬁxed TscanInterval = TscanWindow. Note that wireless data transmission is affected by many physical and environmental conditions, which degrade the behavior of the system in real-life conditions. 4. Experimental and Simulation Validation of the Analytical Model This is the reason why the experiments, simulations and analysis are performed in a controlled scenario without interferences and with the best channel conditions. Once we verify that the numerical results from the mathematical model closely match those obtained in the simulation and testbed measurements, extensive experiments could be performed with BLE devices in several channel/interference conditions, which could be easily included in the mathematical model through the appropriate BLER values. Related to the advertisers, several previous works [17] focus on the performance impact in terms of the tradeoff between consumption and discovery latency, connected with the relationship between TadvInterval and TscanWindow, within a scenario where TscanWindow < TscanInterval. However, in our case, given that continuous scanning is assumed, the main objective is to verify how the real peculiarities of scanning patterns of the BLE chipsets affect the discovery process and, even, notably modify the ideal expected performance. Regarding the TadvInterval duration inﬂuence, it is evident that larger TadvInterval values result in lower energy consumption on the advertiser side and lower collision probability at the scanner. Nevertheless, the mean number of possible detections on a short window of opportunity decreases or even becomes zero. As we said above, in some scenarios, redundancy by repetition could be beneﬁcial. Thus, we want to quantify for an increased number of simultaneous advertisers the impact of TadvInterval in the non-detection probability, the discovery delay and the mean number of detections. About ADV_PDUs sizes, it seems also clear that shorter ADV_PDUs will result in lower collision probabilities, whereas longer ADV_PDUs allow one to extend the capabilities of the system; for instance, monitoring physiological parameters in a sporting event (for example, averaged health parameters, such as temperature, pulse/heart rate, oxygen saturation), that only require a few bytes in order to record global statistics or setup health alarms concerning a speciﬁc participant. Thus, it is also interesting to quantify the discovery performance connected with the ADV_PDU size for several TadvInterval settings. Summarizing, the objectives of the evaluations performed in this section are: Summarizing, the objectives of the evaluations performed in this section are: • To validate the characterization performed in Section 2 of the non-idealities present on the scanning process by comparing simulations and experiment tests with an increasing number of BLE advertisers and different parameter settings. 4. Experimental and Simulation Validation of the Analytical Model • To validate the characterization performed in Section 2 of the non-idealities present on the scanning process by comparing simulations and experiment tests with an increasing number of BLE advertisers and different parameter settings. • To analyze how close the numerical results from the mathematical model match those obtained in the simulations and experimental tests for any parameter values speciﬁed in the standard and also considering the real chipset behavior. This ratiﬁes the potential beneﬁts of using a simple analytical model in order to easily obtain an upper bound of discovery capacities in a potential application scenario. pp • To carry out an analysis in ideal channel conditions that allows one to evaluate the non-detection probability, delay, etc., as the number of BLE devices increases for several parameter settings and real chipsets. pp • To carry out an analysis in ideal channel conditions that allows one to evaluate the non-detection probability, delay, etc., as the number of BLE devices increases for several parameter settings and real chipsets. 16 of 23 16 of 23 Sensors 2017, 17, 499 4.1. Testbed Conditions We performed a series of tests in the scenario following the schema depicted in Figure 2. All of the devices, up to 14 advertisers and one scanner, were placed inside an RF-shield box, always with the same arrangement; see Figure 8. The scanner was connected to a laptop where we performed the raw capture of the data using Tshark. Figure 8. Experimental testbed. Figure 8. Experimental testbed. Tshark is the command line tool of Wireshark [22], a common network protocol analyzer usually employed to audit local area networks, but which can also be used to analyze frames captured through a Bluetooth interface. We then processed the raw data, ﬁltering by interface and advertiser in order to extract different statistics and calculate the non-detection probability and time between consecutive detections. With this conﬁguration, the scanners were programmed with a 500-ms value for both the scan window and scan interval. The advertisers had a wider variation of conﬁgurations. The advertisement interval, TadvInterval, and the size of the advertisement data are set according to the evaluation conditions considered in Section 4.3. Once the number of selected devices were programmed, we double checked their conﬁguration with the android application of Nordic Semiconductor, nRF Connect for Mobile [23]. Finally, we closed the RF-shield box and proceeded with the measurements. The transmission power of advertising devices could be conﬁgured in a range between −40 dBm and 4 dBm. For this range of values, we checked that there was no impact on the BLER in our scenario because the propagation losses are very low inside the RF-shield box. In order to observe some variation on the BLER, it was necessary to insert an extra attenuation shielding the advertising device. The ﬁnal employed value for all devices was 4 dBm. We captured data during three hours for each parameter combination set. 4.3. Performance Results The evaluation has been conducted using the setting as listed in Table 5. Figure 9 compares the analytical model (Model) and the simulation results (Sim) obtained for the ideal implementation of the standard to those obtained when real scanning devices are assumed, including in the last case, the results obtained in the experimental test (Exp) for a number of NBLE devices up to 14. The transmission power of advertising devices was set to 4 dBm, and it has been veriﬁed that in this case and even when power is set to −40 dBm, the non-detection probability for only one advertising device corresponds to PscanGap noDetect. Therefore, BLER effects are almost negligible and enable the comparison of results between the model and simulations in ideal channel conditions. Figure 9a1–a3 shows the non-detection probability for all of these cases, as the number of advertiser BLEs increases from two to 14 for several Tadv values (176 µs, 248 µs and 376 µs) and for different TadvInterval intervals (100 ms in Figure 9a1, 300 ms in Figure 9a2 and 500 ms in Figure 9a3 with TadvDelayMax = 10 ms). From these ﬁgures, it is observed that the numerical results from the mathematical model match with the simulation ones for both ideal and real implementations and for any parameter setting. At the same time, it seems clear that model and simulations perfectly match the experimental results, thus validating the characterization of the scanner process in the actual chipsets. We can see how non-detection probability increases with the number of simultaneous advertising devices under the scanner coverage and also with higher Tadv and TadvInterval values. By comparing the results, we can observe that differences between the ideal standard and the actual implementations are quite signiﬁcant. It is evident that the assumption of the ideal case implies a clear underestimation of non-detection probability. On the other hand, differences between chipsets from different manufacturers need to be taken into account. In this particular case, the Type 1 scanning device provides better results than the Type 2 up to NBLE = 8 when TadvInterval is equal to 100 ms. This advantage is maintained up to NBLE = 21 when TadvInterval is equal to 300 ms for Tadv = 376 µs or up to 23 for Tadv = 176 µs. 4.2. Simulation In order to validate both the analytical model and experimental results, we developed a BLE simulation framework in C++ that fully complies with the BLE advertisement speciﬁcation. We also included two scanner conﬁgurations with the scan patterns identiﬁed in Section 2. The simulations were conducted without any of the simpliﬁcations or assumptions used to derive the analytical model. In order to obtain the desired statistics, the discovery process was monitored in a number 17 of 23 Sensors 2017, 17, 499 of coverage time intervals. In the simulation, the initial instant for each coverage time interval (TcovWindow) was randomly chosen at any continuous time between [0, TscanWindow], and in the same way, the starting transmission time of each advertising device was independently generated between [0, TadvInterval + TadvDelayMax]. A number up to 50,000 coverage time intervals was considered when NBLE is lower than 20 and up to 10,000 for a higher number of devices. Simulation allows including errors due to channel or interference conditions. However, the results are obtained without considering any reception failure of ADV_PDUs (i.e., BLER = 0%) in order to meet experimental testbed conditions. 4.3. Performance Results Finally, the Type 1 device is superior up to NBLE = 35 when TadvInterval is equal to 500 ms with Tadv = 376 µs and up to 37 for Tadv = 176 µs. Table 5. Parameters used in the evaluation. General Parameters Real Scanner Service Parameters Parameter Values Parameter Value Type 1 Pattern Type 2 Pattern Tadv 176 µs, 248 µs, 376 µs Tf qChgGap 1.1 ms 16.05 ms TadvInterval 100 ms, 300 ms, 500 ms TinterFqChgGap - 300 µs τadvDelay Uni f orm(0, TadvDelayMax) TgapInt1 - 16.82 ms TadvDelayMax 10 ms TgapInt2 - 4.3 ms TscanInterval 500 ms τdecodGap Uni f orm(TminDecodGap, TmaxDecodGap) TscanWindow 500 ms TminDecodGap 350 µs 194 µs NBLE 2 to 200 TmaxDecodGap 1.6 ms 194 µs TcovWindow 5 s Table 5. Parameters used in the evaluation. Table 5. Parameters used in the evaluation. Sensors 2017, 17, 499 18 of 23 176s Exp 248s Exp 376s Exp 176s Sim 248s Sim 376s Sim 176s Model 248s Model 376s Model 0 0.05 0.1 0.15 0.2 2 4 6 8 10 12 14 Ideal devices Real devices type 2 Real devices type 1 Non-detection probability Number of devices TadvInterval=100ms (a1) 0.105 0.11 0.115 0.12 0.125 0.13 2 4 6 8 10 12 14 Mean time between consecutive detections (s) Number of devices Ideal devices Real devices type 2 Real devices type 1 TadvInterval=100ms (b1) 0 0.02 0.04 0.06 0.08 0.1 2 4 6 8 10 12 14 Ideal devices Real devices type 2 Real devices type 1 Non-detection probability Number of devices TadvInterval=300ms (a2) 0.305 0.31 0.315 0.32 0.325 0.33 0.335 0.34 2 4 6 8 10 12 14 Mean time between consecutive detections (s) Number of devices Ideal devices Real devices type 2 Real devices type 1 TadvInterval=300ms (b2) 0 0.02 0.04 0.06 0.08 0.1 2 4 6 8 10 12 14 Ideal devices Real devices type 2 Real devices type 1 Non-detection probability Number of devices TadvInterval=500ms (a3) 0.5 0.51 0.52 0.53 0.54 0.55 2 4 6 8 10 12 14 TadvInterval=500ms Mean time between consecutive detections (s) Number of devices Real devices type 2 Real devices type 1 Ideal devices (b3) Figure 9. 4.3. Performance Results Non-detection probability (a1–a3) and mean time between consecutive detections in seconds (b1–b3) as the number of BLE advertisers increases, for several Tadv values (176 µs, 248 µs and 376 µs) and for different TadvInterval intervals (100 ms, 300 ms and 500 ms) with TadvDelayMax = 10 ms. Comparison among experimental measurements, simulation and the analytical model. Sim Sim Sim 176s Model 248s Model 376s Model 0.105 0.11 0.115 0.12 0.125 0.13 2 4 6 8 10 12 14 Mean time between consecutive detections (s) Number of devices Ideal devices Real devices type 2 Real devices type 1 TadvInterval=100ms (b1) 0 0.02 0.04 0.06 0.08 0.1 2 4 6 8 10 12 14 Ideal devices Real devices type 2 Real devices type 1 Non-detection probability Number of devices TadvInterval=300ms (a2) 0.305 0.31 0.315 0.32 0.325 0.33 0.335 0.34 2 4 6 8 10 12 14 Mean time between consecutive detections (s) Number of devices Ideal devices Real devices type 2 Real devices type 1 TadvInterval=300ms (b2) (b2) (a ) 0 0.02 0.04 0.06 0.08 0.1 2 4 6 8 10 12 14 Ideal devices Real devices type 2 Real devices type 1 Non-detection probability Number of devices TadvInterval=500ms (a3) ( ) 0.5 0.51 0.52 0.53 0.54 0.55 2 4 6 8 10 12 14 TadvInterval=500ms Mean time between consecutive detections (s) Number of devices Real devices type 2 Real devices type 1 Ideal devices (b3) (a3) (b3) Figure 9. Non-detection probability (a1–a3) and mean time between consecutive detections in seconds (b1–b3) as the number of BLE advertisers increases, for several Tadv values (176 µs, 248 µs and 376 µs) and for different TadvInterval intervals (100 ms, 300 ms and 500 ms) with TadvDelayMax = 10 ms. Comparison among experimental measurements, simulation and the analytical model. 4.3. Performance Results 19 of 23 Sensors 2017, 17, 499 SIMULATION 176s-100ms 176s-300ms 176s-500ms 248s-100ms 248s-300ms 248s-500ms 376s-100ms 376s-300ms 376s-500ms 176s-100ms 176s-300ms 176s-500ms 248s-100ms 248s-300ms 248s-500ms 376s-100ms 376s-300ms 376s-500ms ANALYTICAL MODEL 0 0.2 0.4 0.6 0.8 1 20 40 60 80 100 120 140 160 180 200 Non-detection probability Number of devices Ideal devices Tadvincreasing (a1) T advInterval=300ms T advInterval=100ms 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 20 40 60 80 100 120 140 160 180 200 Ideal devices Mean time between consecutive detections (s) Number of devices Tadvincreasing T advInterval=500ms (b1) 5 10 15 20 25 30 35 40 45 20 40 60 80 100 120 140 160 180 200 Mean number of detections under coverage Number of devices Ideal devices Tadvincreasing (c1) 0 0.2 0.4 0.6 0.8 1 20 40 60 80 100 120 140 160 180 200 Non detection probability Number of devices Real devices type 1 Tadvincreasing (a2) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 20 40 60 80 100 120 140 160 180 200 Mean time between consecutive detections (s) Number of devices Real devices type 1 Tadvincreasing T advInterval=300ms T advInterval=100ms T advInterval=500ms (b2) 5 10 15 20 25 30 35 40 45 20 40 60 80 100 120 140 160 180 200 Mean number of detections under coverage Number of devices Real Devices Type 1 Tadvincreasing (c2) 0 0.2 0.4 0.6 0.8 1 20 40 60 80 100 120 140 160 180 200 Real devices type 2 Non-detection probability Number of devices Tadvincreasing (a3) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 20 40 60 80 100 120 140 160 180 200 Real devices type 2 Mean time between consecutive detections (s) Number of devices Tadvincreasing T advInterval=300ms T advInterval=100ms T advInterval=500ms (b3) 5 10 15 20 25 30 35 40 45 20 40 60 80 100 120 140 160 180 200 Real devices type 2 Number of devices Tadvincreasing Mean number of detections under coverage (c3) Figure 10. Non-detection probability (a1–a3), mean time between consecutive detections in seconds (b1–b3) and mean number of detections under coverage (TcovWindow = 5 s) (c1–c3), as the number of advertisers increase, for several Tadv values (176 µs, 248 µs and 376 µs) and for different TadvInterval intervals (100 ms, 300 ms and 500 ms) with TadvDelayMax = 10 ms. 4.3. Performance Results Comparison between simulation and the analytical model for ideal (a1,b1,c1), Type 1 real devices (a2,b2,c2) and Type 2 real devices (a3,b3,c3). T advInterval=300ms T advInterval=100ms 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 20 40 60 80 100 120 140 160 180 200 Ideal devices Mean time between consecutive detections (s) Number of devices Tadvincreasing T advInterval=500ms (b1) 0 0.2 0.4 0.6 0.8 1 20 40 60 80 100 120 140 160 180 200 Non-detection probability Number of devices Ideal devices Tadvincreasing (a1) 5 10 15 20 25 30 35 40 45 20 40 60 80 100 120 140 160 180 200 Mean number of detections under coverage Number of devices Ideal devices Tadvincreasing (b1) (c1) 5 10 15 20 25 30 35 40 45 20 40 60 80 100 120 140 160 180 200 Mean number of detections under coverage Number of devices Real Devices Type 1 Tadvincreasing ( ) (a1) (c1) (b1) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 20 40 60 80 100 120 140 160 180 200 Mean time between consecutive detections (s) Number of devices Real devices type 1 Tadvincreasing T advInterval=300ms T advInterval=100ms T advInterval=500ms (b2) ( ) 0 0.2 0.4 0.6 0.8 1 20 40 60 80 100 120 140 160 180 200 Non detection probability Number of devices Real devices type 1 Tadvincreasing (a2) Real Devices Type 1 (c2) 5 10 15 20 25 30 35 40 45 20 40 60 80 100 120 140 160 180 200 Real devices type 2 Number of devices Tadvincreasing Mean number of detections under coverage (c3) (c2) (b2) (a2) (a2) 0 0.2 0.4 0.6 0.8 1 20 40 60 80 100 120 140 160 180 200 Real devices type 2 Non-detection probability Number of devices Tadvincreasing (a3) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 20 40 60 80 100 120 140 160 180 200 Real devices type 2 Mean time between consecutive detections (s) Number of devices Tadvincreasing T advInterval=300ms T advInterval=100ms T advInterval=500ms (b3) Real devices type 2 (b3) (c3) (a3) Figure 10. 4.3. Performance Results In this case, only simulation and mathematical results are illustrated related to the non-detection probability (Figure 10a1–a3), the mean time between consecutive detections (Figure 10b1–b3) and the mean number of detections under coverage (Figure 10c1–c3), as the number of advertisers increases, for several Tadv values (176 µs, 248 µs and 376 µs) and for different TadvInterval with TadvDelayMax = 10 ms. The mean number of detections under coverage evaluation only intends to be illustrative. Discovery capacity needs to be evaluated for each potential application, being TcovWindow particularized to the expected value. In this case, as an example, TcovWindow is set to 5 s. p In order to facilitate the visualization of results, unlike Figure 9, Figure 10 compares separately simulation and the analytical model for ideal, Type 1 scanning devices and Type 2 scanning devices. We show that mathematical curves almost coincide with the simulation results for the entire range of parameters and devices. Differences between actual devices are signiﬁcant. As anticipated before, for a larger number of advertising devices, Type 2 scanning devices clearly offer better results. Note that, when a few advertisers are considered, the impact of the additional micro-gap scans exceeds that caused by gaps due to decoding. However, as long as the number of advertisers increases, decoding interruptions are the most challenging issue in the successful detection probability. In this case, mean τdecodGap is clearly higher in Type 1 devices. Concerning the averaged detection delay, in contrast to Figure 9, in Figure 10b1–b3, we clearly check (see NBLE = 200) that the averaged detection delay is higher as long as decreases, as expected. However, shorter TadvEvent remains to be desirable to achieve a lower mean time between consecutive detections and, thus, a higher number of detections in a TcovWindow. Concerning this last parameter (see Figure 10c1–c3), we can see that using this simple discovery process, the number of potential detections is high enough even for a large number of simultaneous devices, providing a high margin for repetitions in order to combat real propagation and interference conditions with non-negligible BLER. Figure 11a shows the Cumulative Density Function (CDF) of the number of detections for several Tadv = 176 µs, Tadv = 376 µs with TadvInterval = 100 ms and TadvDelayMax = 10 ms, when there are NBLE = 200 BLE advertisers. 4.3. Performance Results Non-detection probability (a1–a3), mean time between consecutive detections in seconds (b1–b3) and mean number of detections under coverage (TcovWindow = 5 s) (c1–c3), as the number of advertisers increase, for several Tadv values (176 µs, 248 µs and 376 µs) and for different TadvInterval intervals (100 ms, 300 ms and 500 ms) with TadvDelayMax = 10 ms. Comparison between simulation and the analytical model for ideal (a1,b1,c1), Type 1 real devices (a2,b2,c2) and Type 2 real devices (a3,b3,c3). However, as is shown in Figure 10, the Type 2 scanning device is a better choice when a high number of advertising devices coexist. In similar conditions, the mean time between consecutive detections is shown in Figure 9b1–b3. This parameter has been chosen instead of the average detection delay because this statistic can be directly obtained by processing the data from experimental measurements without any kind of assumption. We can compare approximately the average detection delay only by subtracting TadvEvent from the mean time between advertisements. As shown in the ﬁgures, results from the mathematical model, simulation and experimental tests match again for both ideal and real implementations for any parameter setting. Focusing on the ideal standard Sensors 2017, 17, 499 20 of 23 implementation, it is easy to check that, as expected, the average detection delay is clearly higher as long as TadvInterval decreases due to the higher non-detection probability. Comparing results for NBLE = 14 and Tadv = 376 µs, they are almost similar. For instance, for ideal devices, the average detection delay is 10.28 ms when TadvInterval = 100 ms (in this case, TadvEvent = 105 ms), 9.94 ms when TadvInterval = 300 ms (TadvEvent = 305 ms) and 9.87 ms when TadvInterval = 500 ms ( TadvEvent = 505 ms). The absolute values of non-detection probability are not high enough to have a notable impact on this parameter. However, differences in the mean time between consecutive detections will have a great impact in the number of potential detections in a ﬁxed coverage time interval (TcovWindow), being a valuable parameter for the intended applications. Figure 10 extends the comparison for a larger number of advertising devices, up to 200. 4.3. Performance Results We can see that, even in the worst case (real device Type 1 and Tadv = 376 µs), less than 7% of the advertisers are detected three or less times. In this same case, the probability of zero detections of a device is 0.000692. Using this probability, we can theoretically derive in a simple way the probability that not all of the devices (200) are detected in TcovWindow, using the expression 1 −(1 −0.000692)200. This probability is around 13%, which agrees with the result obtained by the simulation, as shown in Table 6. Nevertheless, in most of the cases, non-detection affects only one or two devices, as the non-detection probability of a BLE in TcovWindow is very low. In a similar way, Figure 11b shows the CDF of the number of detections for Tadv = 176 µs, Tadv = 376 µs with TadvInterval = 500 ms and TadvDelayMax = 10 ms, when there are NBLE = 200 BLE advertisers. Now, in the worst case (Type 1 real device and Tadv = 376 µs), less than 13.4% of the advertisers are detected three or less times. Table 6 shows the simulation results concerning the probability that not all of the devices (200) are detected in the TcovWindow for Tadv = 176 µs, Tadv = 376 µs with TadvInterval = 100 ms and TadvInterval = 500 ms when TadvDelayMax = 10 ms. Sensors 2017, 17, 499 21 of 23 21 of 23 0 0.2 0.4 0.6 0.8 1 0 5 10 15 20 25 30 35 Number of detections CDF of number of detections under coverage T advInterval=100ms N BLE=200 (a) 0 0.2 0.4 0.6 0.8 1 0 2 4 6 8 10 CDF of number of detections under coverage Number of detections T advInterval=500ms N BLE=200 (b) Figure 11. CDF of the number of detections under coverage (TcovWindow = 5 s) for several Tadv values (Tadv = 176 µs, Tadv = 376 µs) when TadvInterval = 100 ms (a); and TadvInterval = 500 ms (b) with TadvDelayMax = 10 ms and when there are NBLE = 200 BLE advertisers. Comparison between simulation results for ideal devices, Type 1 real devices and Type 2 real devices. 4.3. Performance Results 0 0.2 0.4 0.6 0.8 1 0 2 4 6 8 10 CDF of number of detections under coverage Number of detections T advInterval=500ms N BLE=200 (b) 0 0.2 0.4 0.6 0.8 1 0 5 10 15 20 25 30 35 Number of detections CDF of number of detections under coverage T advInterval=100ms N BLE=200 (a) (b) (a) Figure 11. CDF of the number of detections under coverage (TcovWindow = 5 s) for several Tadv values (Tadv = 176 µs, Tadv = 376 µs) when TadvInterval = 100 ms (a); and TadvInterval = 500 ms (b) with TadvDelayMax = 10 ms and when there are NBLE = 200 BLE advertisers. Comparison between simulation results for ideal devices, Type 1 real devices and Type 2 real devices. Table 6. Probability (in %) that not all of the devices (200) are detected in the TcovWindow (5 s) for several Tadv values (Tadv = 176 µs, Tadv = 376 µs) when TadvInterval = 100 ms and TadvInterval = 500 ms with TadvDelayMax = 10 ms and when there are NBLE = 200 BLE advertisers. Comparison between simulation results for ideal devices, Type 1 real devices and Type 2 real devices. TadvInterval Tadv = 176 µs Tadv = 376 µs Ideal Type 1 Type 2 Ideal Type 1 Type 2 100 ms 0 % 0.04 % 0 % 0.2 % 12.96 % 2.52 % 500 ms 0 % 4.94 % 0.54 % 0.3 % 19.69 % 6.48 % Conclusions TadvInterval Tadv = 176 µs Tadv = 376 µs Ideal Type 1 Type 2 Ideal Type 1 Type 2 100 ms 0 % 0.04 % 0 % 0.2 % 12.96 % 2.52 % 500 ms 0 % 4.94 % 0.54 % 0.3 % 19.69 % 6.48 % References 1. Lee, J.S.; Su, Y.W.; Shen, C.C. A Comparative Study of Wireless Protocols: Bluetooth, UWB, ZigBee, and Wi-Fi. In Proceedings of the 33rd Annual Conference of the IEEE Industrial Electronics Society (IECON), Taipei, Taiwan, 5–8 November 2007; pp. 46–51. 1. Lee, J.S.; Su, Y.W.; Shen, C.C. A Comparative Study of Wireless Protocols: Bluetooth, UWB, ZigBee, and Wi-Fi. In Proceedings of the 33rd Annual Conference of the IEEE Industrial Electronics Society (IECON), Taipei, Taiwan, 5–8 November 2007; pp. 46–51. . Haider, K. Uses of Bluetooth Other than Wireless Audio. 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Abbreviations The following abbreviations are used in this manuscript: ADV Advertising BLE Bluetooth Low Energy BLER Block Error Rate CDF Cumulative Density Function GFSK Gaussian Frequency Shift Keying ISM Industrial Scientiﬁc Medical IoT Internet of Things LTE-M LTE Machine to Machine NB-IoT Narrowband IoT PDU Packet Data Unit PHY Physical SoC System on a Chip ADV Advertising BLE Bluetooth Low Energy BLER Block Error Rate CDF Cumulative Density Function GFSK Gaussian Frequency Shift Keying ISM Industrial Scientiﬁc Medical IoT Internet of Things LTE-M LTE Machine to Machine NB-IoT Narrowband IoT PDU Packet Data Unit PHY Physical SoC System on a Chip 5. Conclusions In this paper, we have experimentally studied the BLE discovery process when a high number of users needs to be discovered in a short period of time. After analyzing several scanning devices from different BLE chipset manufacturers, we have shown that all of them have non-idealities in the scanning process. Even though this aspect is not taken into account in other studies, we have demonstrated that these non-idealities have a severe impact on the performance of the discovery process. We have identiﬁed two main behavior patterns that characterize the diverse actual devices by setting appropriate parameter values. Once these non-idealities have been characterized, an extensive performance analysis for a varying number of devices and feasible parameter combinations concerning the advertising interval and advertising frame size has been conducted. Simulations and experimental measurements for two speciﬁc chipsets have been compared, considering up 14 devices. This study has been extended by simulation for a higher number of devices. It has been observed that the advantages of using a particular scanner chipset over another depend on the number of devices that need to be detected, and they are also conditioned by the selected parameter settings. This work also includes a simple model that meets both the standard implementation and the described peculiarities of the actual implementation on the chipsets. The results from the mathematical model, the simulation results and the experimental measurements of real devices closely match for any feasible parameter values speciﬁed in the standard and for any number of simultaneous advertising devices under a scanner coverage. That is, the model can be useful in order to easily obtain an upper bound for the discovery capacity and to select the desirable parameters values according to a particular BLE application. 22 of 23 Sensors 2017, 17, 499 Acknowledgments: This work has been supported in part by the MINECO/FEDER under the projects TEC2014-58341-C4-2-R and TEC2014-60258-C2-2-R. Acknowledgments: This work has been supported in part by the MINECO/FEDER under the projects TEC2014-58341-C4-2-R and TEC2014-60258-C2-2-R. Author Contributions: All authors have contributed in a similar way in all of the sections of the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest. Acknowledgments: This work has been supported in part by the MINECO/FEDER under the projects TEC2014-58341-C4-2-R and TEC2014-60258-C2-2-R. Author Contributions: All authors have contributed in a similar way in all of the sections of the paper. References On the potential of bluetooth low energy technology for vehicular applications. IEEE Commun. Mag. 2015, 53, 267–275. 13. Bronzi, W.; Frank, R.; Castignani, G.; Engel, T. Bluetooth Low Energy performance and robustness analysis for Inter-Vehicular Communications. Ad Hoc Netw. 2015, 37, 76–86. 14. Liu, J.; Chen, C.; Ma, Y. Modeling Neighbor Discovery in Bluetooth Low Energy Networks. IEEE Commun. Lett. 2012, 16, 1439–1441. 23 of 23 Sensors 2017, 17, 499 15. Liu, J.; Chen, C.; Ma, Y.; Xu, Y. Energy Analysis of Device Discovery for Bluetooth Low Energy. 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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/).
https://openalex.org/W4290053048	https://zenodo.org/records/6969413/files/15-22.pdf	Indonesian	null	O'ZBEKISTON DIPLOMATIYASINING TARIXI VA ISTIQBOLLARI	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	2,111	ABSTRACT This article describes in detail the history of diplomacy of the republic of uzbekistan and its future prospects, and provides a detailed analysis of the current work in this area in our country. Keywords: diplomacy, international relations, ambassador, foreign policy, united nations general assembly, us state department. ANNOTATSIYA Ushbu maqolada O‘zbekiston Respublikasi diplomatiyasining tarixi va uning kelajakdagi istiqbollari batafsil yoritilgan bo‘lib, hozirgi kunda ushbu sohada mamlakatimizda olib borilayotgan ishlar atroflicha tahlil qilingan. Kalit so‘zlar: diplomatiya, xalqaro munosabatlar, elchi, tashqi siyosat, Birlashgan Millatlat Tashkiloti Bosh Assambleyasi, AQSh Davlat departamenti. O'ZBEKISTON DIPLOMATIYASINING TARIXI VA ISTIQBOLLARI O'ZBEKISTON DIPLOMATIYASINING TARIXI VA ISTIQBOLLA Moʻydinova Mahfuzaxon Muhammadjonovna Fargʻona viloyati Dangʻara tumani XTB tasarrufidagi 7-umumiy oʻrta taʼlim maktabi Tarix-huquq fani oʻqituvchisi ISSN: 2181-4027_SJIF: 4.995 ISSN: 2181-4027_SJIF: 4.995 “PEDAGOGS” international research journal KIRISH O‘zbekiston Respublikasi mustaqillikka erishgandan so‘ng turli xil sohalar qatori tashqi siyosiy yo‘nalishlarni rivojlantirish bo‘yicha amaliy harakatlarni boshlagan. O‘zbekiston o‘z oldiga nafaqat izchil ichki siyosat yuritishni, balki faol tashqi siyosat yuritishni ham asosiy vazifa qilib qo‘ygan bo‘lib, shu maqsad yo‘lida tizimli ishlar olib bormoqda. Volume-15, Issue-1, August- 2022 www.pedagoglar.uz www.pedagoglar.uz 15 www.pedagoglar.uz ISSN: 2181-4027_SJIF: 4.995 Tarixdan ma'lumki, O‘zbekiston hududida mavjud bo‘lgan ilk davlatlar: Turk xoqonligi, Arab xalifaligi, Temuriylar va uch xonliklar davrida ham diplomatiya davlatning asosiy e'tibor qaratiladigan sohasi bo‘lgan, chunki mustaqillikni mustahkamlash, mamlakatning xavfsizligi, barqarorligi va taraqqiyoti ko‘p jihatdan xorijiy davlatlar bilan olib boriladigan faol tashqi siyosatga bog‘liq. Buyuk ipak yo‘lida joylashgan mamlakatimiz asrlar davomida xorijiy davlatlar bilan iqtisodiy, siyosiy va madaniy munosabatlarni shakllantirib, o‘zaro diplomatik aloqalar olib borgan. Bu jarayon, ayniqsa, buyuk davlat arbobi, Sohibqiron Amir Temur davrida rivoj topgan. Amir Temur o‘z davrining eng qudratli davlatlaridan birining asoschisi, ilm-fan va madaniyat homiysi bo‘lish bilan bilan bir qatorda, o‘z vaqtida Yevropa va Osiyo qit'alarida ro‘y bergan tarixiy o‘zgarishlarga hal qiluvchi ta'sir o‘tkazib, jahon sivilizatsiyasi taraqqiyotiga beqiyos hissa qo‘shgan mohir diplomat ham bo‘lgan. Xorijiy davlatlarning hukmdorlari bilan olib borgan yozishmalarida, diplomatik xatlarida sharq diplomatiyasiga xos etiketning yaqqol sezilib turishi, mamlakatlar o‘rtasidagi sovuqchilik va muammolarni tinch yo‘l bilan oqilona hal qilishga intilishi Amir Temur tashqi siyosatining o‘ziga xosligidan dalolatdir. Shuning uchun ham Temuriylar davri diplomatiyasi o‘tmishdagi xalqaro munosabatlarning eng yorqin sahifalardan biri hisoblanadi. ADABIYOTLAR TAHLILI VA METODOLOGIYA Amir Temur bundan olti asr avvaloq xorijiy davlatlar bilan tashqi aloqalar o‘rnatmay turib, mamlakatning istiqbolga erishib bo‘lmasligini teran anglagan edi. Angliya, Fransiya, Usmonli Turklar, Ispaniya, Xitoy, Hindiston kabi turli mamlakat hukmdorlari bilan amalga oshirgan yozishmalari va diplomatik aloqalari buning yaqqol tasdig‘idir. Yurtimizdan Amir Temurga o‘xshagan mohir diplomat va elchilar ko‘plab yetishib chiqqanligiga qaramay, ularning faoliyati chuqur o‘rganilmagan. Bu haqda davlatimiz rahbari Shavkat Mirziyoyev 2018-yilning 11-yanvar kuni bo‘lib o‘tgan O‘zbekiston Respublikasi Tashqi ishlar vazirligi va mamlakatimizning xorijdagi elchixonalari faoliyatiga bag‘ishlagan nutqida: “Bizning boy madaniyat va qadimiy tarixga ega davlatimizdan o‘z davrining mohir diplomatlari, elchilari yetishib chiqqani Volume-15, Issue-1, August- 2022 16 www.pedagoglar.uz “PEDAGOGS” international research journal “PEDAGOGS” international research journal ISSN: 2181-4027_SJIF: 4.995 www.pedagoglar.uz ISSN: 2181-4027_SJIF: 4.995 ISSN: 2181-4027_SJIF: 4.995 “PEDAGOGS” international research journal to‘g‘risida ko‘plab ma'lumotlar mavjud, ammo ularning hayoti va faoliyati, afsuski, hozirgacha chuqur va atroflicha o‘rganilmagan. Tashqi ishlar vazirligi bunga e'tibor qaratib, ushbu masalaning tarixiy ildizlari, mustaqillik davrida o‘zbek diplomatiyasining shakllanish jarayonlari haqida alohida bir kitob chiqarsa, bu soha xodimlari, avvalo, yosh diplomatlar uchun muhim qo‘llanma bo‘lardi”, – deb ta'kidlab o‘tgan. XIX asrda O‘zbekiston hududida joylashgan davlatlar Rossiya Imperiyasi tomonidan mustamlakaga aylantirilgandan so‘ng mustaqil tashqi siyosat yuritish huquqidan mahrum bo‘lgan, suverenitetini yo‘qotgan, faqatgina markaz manfaatlari uchun xizmat qilishga majbur bo‘lgan mamlakatimiz uchun tashqi siyosat o‘z ahamiyatini yo‘qotgan edi. Ammo xalqimizning asriy orzu-istaklari ro‘yobga chiqib, istiqlolga erishganimizdan so‘ng suveren O‘zbekiston diplomatiyasining dastlabki qadamlari qo‘yildi. Jumladan, O‘zbekiston Respublikasining tashqi siyosatini samarali yo‘lga qo‘yish, Respublikaning siyosiy va ijtimoiy-iqtisodiy rivojlanishi uchun qulay tashqi shart-sharoitni vujudga keltirish, xalqaro munosabatlarda va xorijiy mamlakatlarda O‘zbekiston Respublikasi fuqarolari va davlat manfaatlarini ifodalash va himoya qilish maqsadida Vazirlar Mahkamasining 1992-yil 25-mayda “O‘zbekiston Respublikasi Tashqi ishlar vazirligining faoliyatini tashkil etish masalalari to‘g‘risida”gi qarori qabul qilindi. O‘zbekiston Respublikasining tashqi siyosati 1994-yil mart oyida qabul qilingan “O‘zbekiston Respublikasi Tashqi ishlar vazirligi faoliyatini takomillashtirish to‘g‘risida”gi va 1996-yildagi “O‘zbekiston Respublikasi tashqi siyosiy faoliyatining asosiy prinsiplari to‘g‘risida”gi qarorlar asosida shakllantirildi. Shu tufayli istiqlol yillarida davlatimiz 133 dan ortiq mamlakatlar bilan rasmiy munosabatlar o‘rnatdi, hozirda Toshkentda 45 ta xorijiy davlatlarning elchixonalari, 9 ta konsullik, 11 ta xalqaro tashkilotlar vakolotxonalari faoliyat olib bormoqda. O‘zbekiston Respublikasi Tashqi siyosatining asosiy tamoyillari, prinsiplari, strategik ustuvor yo‘nalishlari, maqsad va vazifalari 2012-yil sentabrda qabul qilingan “Tashqi siyosiy faoliyat konsepsiyasi”da o‘z aksini topgan. Bu konsepsiya O‘zbekiston milliy manfaatlarini ilgari suradigan ustuvor yo‘nalishlarni belgilab beradigan qarashlarning yaxlit tizimidir. Volume-15, Issue-1, August- 2022 17 “PEDAGOGS” international research journal ISSN: 2181-4027_SJIF: 4.995 “PEDAGOGS” international research journal mavzusida 500 nafardan ortiq delegat ishtirok etgan anjuman bo‘lib o‘tdi. Davlatimiz rahbari Markaziy Osiyo davlatlari bilan do‘stona, yaqin qo‘shnichilik va o‘zaro manfaatli aloqalarni mustahkamlash va rivojlantirishni asosiy tashqi siyosiy yo‘nalish sifatida belgilab berdi. Tariximizning, dinimizning, madaniyatimizning, tilimizning birligi, tomirlarimizning tutashib ketganligi hozirgi tahlikali zamonda bu mamlakatlar xalqlarini yanada yaqinlashtirish kerakligini har qachongidan ham ko‘proq taqozo eta boshladi. Prezident Shavkat Mirziyoyev tashabbusi bilan 2017-yilda O‘zbekistonning qo‘shni davlatlar bilan munosabatlarida yangi davr boshlandi. Shu yilning o‘zida Turkmaniston, Qozog‘iston va Qirg‘iziston davlatlariga davlat tashriflar amalga oshirildi. Davlat rahbarlari o‘rtasida ochiq va ishonchli siyosiy muloqotni mustahkamlash natijasida mintaqadagi qo‘shnichilik munosabatlarida 2016-yilgacha mavjud bo‘lgan ko‘plab murakkab muammolarga yechim topildi. O‘zbekistonning zamonaviy tashqi siyosati faol, tashabbuskor va pragmatik tashqi siyosiy kurs olib borishni hamda yuzaga kelayotgan xavf-xatarlarga o‘z vaqtida javob berishni talab etadigan XXI asrning o‘ta shiddat bilan o‘zgarib borayotgan xalqaro-siyosiy voqeliklarini inobatga olgan holda qurilmoqda. Bular O‘zbekistonning jahon hamjamiyati bilan mustahkam aloqada bo‘lganligidan, har tomonlama manfaatli hamkorlikni yanada chuqurlashtirib borayotganidan dalolat beradi. Xalqaro hamkorliklar doirasida qabul qilingan hujjat va kelishuvlarni o‘z vaqtida to‘liq bajarish mavzusida 500 nafardan ortiq delegat ishtirok etgan anjuman bo‘lib o‘tdi. Davlatimiz rahbari Markaziy Osiyo davlatlari bilan do‘stona, yaqin qo‘shnichilik va o‘zaro manfaatli aloqalarni mustahkamlash va rivojlantirishni asosiy tashqi siyosiy yo‘nalish sifatida belgilab berdi. Tariximizning, dinimizning, madaniyatimizning, tilimizning birligi, tomirlarimizning tutashib ketganligi hozirgi tahlikali zamonda bu mamlakatlar xalqlarini yanada yaqinlashtirish kerakligini har qachongidan ham ko‘proq taqozo eta boshladi. Prezident Shavkat Mirziyoyev tashabbusi bilan 2017-yilda O‘zbekistonning qo‘shni davlatlar bilan munosabatlarida yangi davr boshlandi. Shu yilning o‘zida Turkmaniston, Qozog‘iston va Qirg‘iziston davlatlariga davlat tashriflar amalga oshirildi. Davlat rahbarlari o‘rtasida ochiq va ishonchli siyosiy muloqotni mustahkamlash natijasida mintaqadagi qo‘shnichilik munosabatlarida 2016-yilgacha mavjud bo‘lgan ko‘plab murakkab muammolarga yechim topildi. O‘zbekistonning zamonaviy tashqi siyosati faol, tashabbuskor va pragmatik tashqi siyosiy kurs olib borishni hamda yuzaga kelayotgan xavf-xatarlarga o‘z vaqtida javob berishni talab etadigan XXI asrning o‘ta shiddat bilan o‘zgarib borayotgan xalqaro-siyosiy voqeliklarini inobatga olgan holda qurilmoqda. Bular O‘zbekistonning jahon hamjamiyati bilan mustahkam aloqada bo‘lganligidan, har tomonlama manfaatli hamkorlikni yanada chuqurlashtirib borayotganidan dalolat beradi. Xalqaro hamkorliklar doirasida qabul qilingan hujjat va kelishuvlarni o‘z vaqtida to‘liq bajarish Dunyoda kechayotgan murakkab geosiyosiy jarayonlar, koronavirus pandemiyasi va butunjahon iqtisodiy inqirozi sharoitida O‘zbekiston ochiq va pragmatik faol tashqi siyosat olib bormoqda. Xalqaro maydondagi qat'iy sa'y- harakatlarimiz natijasida yurtimizning nufuzi va obro‘-e'tibori tobora yuksalmoqda. Dunyo mamlakatlari orasida yurtimiz o‘z o‘rniga ega bo‘lmoqda. Jumladan, mamlakatimiz o‘z tarixida birinchi marta BMTning Inson huquqlari bo‘yicha kengashiga a'zo etib saylandi. www.pedagoglar.uz MUHOKAMA VA NATIJALAR O‘zbekiston Respublikasi ko‘p tomonlama hamkorlik aloqalarini rivojlantirish uchun faol va tinchlikparvar tashqi siyosat yuritgan. Masalan, 1992-yil 2-mart kuni O‘zbekiston Respublikasi Birlashgan Millatlar Tashkilotining teng huquqli a'zosiga aylanib, xalqaro minbarlarda mamlakat nomidan turli xil takliflar ilgari surishni boshlagan. Jumladan, butun dunyoda tinchlik va barqarorlikni mustahkamlash, Afg‘onistonda tinchlik o‘rnatish, xalqlarning ijtimoiy-iqtisodiy taraqqiyotiga ko‘maklashish, yadro quroli tarqalishining oldini olish bo‘yicha bildirilgan takliflar jahon hamjamiyatini birlashtirishga intilayotgan BMT tomonidan qizg‘in qo‘llab- quvvatlandi. O‘zbekiston BMTning bir qator ixtisoslashtirilgan muassasalari, jumladan, Xalqaro valyuta fondi (XVF), Jahon banki, BMTning Yevropa bo‘yicha iqtisodiy komissiyasi (YeIK), Jahon savdo tashkiloti (JST) kabi tashkilotlar bilan keng ko‘lamli hamkorlik aloqalarini yo‘lga qo‘ydi. Shuningdek, O‘zbekiston Respublikasi BMT doirasidagi ixtisoslashgan muassasalar: Xalqaro bolalar jamg‘armasi (YuNISEF), Jahon sog‘liqni saqlash tashkiloti, Xalqaro Olimpiya qo‘mitasi, Xalqaro mehnat tashkiloti va boshqa tashkilotlarga ham a'zo bo‘ldi. Xalqaro tashkilotlar bilan hamkorlik qilish orqali tashqi siyosatda yuqori reytinglarga ega bo‘lish davomida tizimda uchragan ba'zi kamchiliklar tufayli mamlakatimizda bir muddat tashqi aloqalar sezilarli darajada sustlashib qoldi. Ayniqsa, bu holat 2000-yillar o‘rtalaridan boshlab sezila boshladi. Xorijiy davlatlar bilan, eng asosiysi, bizga chegaradosh bo‘lgan mamlakatlar bilan turli sohalardagi hamkorlikning sustlashishi natijasida tizimda ko‘plab muammolar paydo bo‘ldi. Biroq, 2016-yilning so‘nggi choragida prezident etib saylangan Shavkat Mirziyoyev bu holatni chuqur anglagan xolda turli sohalar qatori tashqi siyosat tizimini ham tanqidiy qayta ko‘rib chiqish va uni kuchaytirish ustuvor vazifa ekanligiga alohida e'tibor qaratdi. Bu borada bir qancha amaliy ishlar olib borildi. Jumladan, mamlakatimiz rahbari 2018-yilning yanvar oyida mamlakatimiz tarixida ilk bor O‘zbekistonning xorijiy davlatlardagi elchilari bilan ochiq onlayn muloqot o‘tkazdi. Bundan tashqari, 2017-yilda Samarqand shahrida “Markaziy Osiyo: yagona tarix va umumiy kelajak, taraqqiyot va barqaror rivojlanish yo‘lidagi hamkorlik” Volume-15, Issue-1, August- 2022 18 ISSN: 2181-4027_SJIF: 4.995 ISSN: 2181-4027_SJIF: 4.995 “PEDAGOGS” international research journal ISSN: 2181-4027_SJIF: 4.995 Yaqinda AQSh Davlat departamenti tomonidan O‘zbekiston diniy erkinlik sohasidagi “maxsus kuzatuvdagi davlatlar” ro‘yxatidan chiqarilgani ham keng ko‘lamli demokratik islohotlarimizning e'tirofidir. Shavkat Volume-15, Issue-1, August- 2022 19 “PEDAGOGS” international research journal Mirziyoyevning 2017-yil 19-sentabrda Birlashgan Millatlar Tashkiloti Bosh Assambleyasining oliy minbarida ilgari surgan global va mintaqaviy tashabbuslari dunyo hamjamiyati tomonidan katta qiziqish bilan qabul qilinib, qo‘llab-quvvatlandi. 2020-yil 29-dekabrda bo‘lib o‘tgan O‘zbekiston Respublikasi Prezidenti Shavkat Mirziyoyevning Oliy Majlisga Murojaatnomasida 2021-yilda mamlakatimizning tashqi siyosatini amalga oshirishda quyidagi vazifalarga ustuvor ahamiyat berilishishi aytib o‘tildi: Birinchidan, davlatimiz olib borayotgan ochiq, pragmatik va amaliy tashqi siyosatni inobatga olib hamda uzoq muddatli strategik maqsadlarimizdan kelib chiqqan holda, O‘zbekiston Respublikasining Tashqi siyosiy faoliyat konsepsiyasi takomillashtiriladi. Ikkinchidan, tashqi siyosatimizning ustuvor yo‘nalishlaridan bo‘lgan Markaziy Osiyo davlatlari bilan ko‘p asrlik do‘stlik va yaxshi qo‘shnichilik, strategik sheriklik va o‘zaro ishonch ruhidagi aloqalarimizni yanada mustahkamlashga alohida e'tibor qaratiladi. Murakkab pandemiya sharoiti biz bir-birimizga qanchalik bog‘liq va kerak ekanimizni yana bir bor yaqqol namoyon etdi. Bu ijobiy jarayonlarni chuqur o‘rganish va yanada rivojlantirish maqsadida 2020-yilda Toshkent shahrida Markaziy Osiyo xalqaro instituti tashkil etildi. 2021-yilda Toshkentda mintaqamizning Janubiy Osiyo bilan munosabatlariga doir yuqori darajadagi xalqaro konferensiya o‘tkaziladi. Shular qatorida Xiva shahrida “Markaziy Osiyo jahon sivilizatsiyalari chorrahasida” xalqaro anjumani YuNESKO bilan hamkorlikda o‘tkaziladi. Uchinchidan, O‘zbekiston “Qo‘shning tinch-sen tinch” tamoyili asosida qo‘shni Afg‘oniston zaminida tinchlik o‘rnatilishi uchun tizimli ishlar olib borgan va bu ezgu yo‘lda amaliy yordamni bundan keyin ham ayamaydi. Hozirgi kunda Markaziy Osiyoni Hind okeani bilan bog‘laydigan Trans-afg‘on transport yo‘lagini barpo etish borasida dastlabki amaliy qadamlar tashlandi. Bu loyihaning ro‘yobga chiqarilishi butun mintaqamizda barqarorlik va iqtisodiy o‘sishni ta'minlashga beqiyos xizmat qiladi. Volume-15, Issue-1, August- 2022 www.pedagoglar.uz www.pedagoglar.uz 20 “PEDAGOGS” international research journal “PEDAGOGS” international research journal ISSN: 2181-4027_SJIF: 4.995 ISSN: 2181-4027_SJIF: 4.995 ISSN: 2181-4027_SJIF: 4.995 To‘rtinchidan, Birlashgan Millatlar Tashkiloti, boshqa nufuzli xalqaro va mintaqaviy tuzilmalar bilan yaqin hamkorlikni rivojlantirish masalalari doimo e'tiborimiz markazida bo‘ladi. 2020-yilda mamlakatimiz ilk bor Mustaqil Davlatlar Hamdo‘stligi tashkilotiga raislik qildi va pandemiyaga qaramasdan, ko‘zda tutilgan 60 dan ziyod barcha xalqaro tadbirlar muvaffaqiyatli o‘tkazildi, 70 ga yaqin muhim hujjatlar qabul qilindi. 2021-2022-yillarda O‘zbekistonning Shanxay hamkorlik tashkilotiga raislik qilishi bo‘yicha ham jiddiy tayyorgarliklar boshlandi. Beshinchidan, inson huquqlarini ta'minlash, so‘z va diniy e'tiqod erkinligi bo‘yicha erishayotgan yutuqlarimizni tegishli xalqaro tashkilotlar bilan hamkorlikda izchil mustahkamlab borish va kelgusi yilda Birlashgan Millatlar Tashkiloti shafeligida “Inson huquqlari bo‘yicha ta'lim” global forumi, yoshlar huquqlariga bag‘ishlangan Butunjahon yoshlar anjumani, diniy erkinlik masalalari bo‘yicha mintaqaviy konferensiyalarni o‘tkazish maqsad qilingan. Oltinchidan, Shavkat Mirziyoyev dunyoning ko‘plab mamlakatlaridagi vatandoshlarimizni qo‘llab-quvvatlash va ular bilan aloqalarimizni yanada mustahkamlash maqsadida “Vatandoshlar” jamg‘armasini tuzishni taklif etdi. Yettinchidan, jamiyatda millatlararo totuvlik va bag‘rikenglik muhitini mustahkamlashga qaratilgan ishlarimizni sifat jihatdan yangi bosqichga olib chiqish kerakligini ta'kidladi. Ma'lumki, Birlashgan Millatlar Tashkiloti Bosh Assambleyasining rezolyutsiyasi bilan har yili 30 iyul - Xalqaro do‘stlik kuni sifatida keng nishonlanadi. Shu bois davlatimiz rahbari ushbu sanani O‘zbekistonda “Xalqlar do‘stligi kuni” deb belgilashni taklif etdi. Sakkizinchidan, mintaqamizda, butun dunyoda ekologik vaziyatning tobora yomonlashib borayotgani bizni jiddiy tashvishga solayotgani, qo‘shni davlatlar va jahon jamoatchiligi bilan birgalikda Orol dengizi halokati oqibatida yuzaga kelgan ekologik fojialar ta'sirini yumshatishga qaratilgan harakatlarni qat'iy davom ettirishimiz zarurligi aytib o‘tildi. Volume-15, Issue-1, August- 2022 www.pedagoglar.uz 21 “PEDAGOGS” international research journal XULOSA Xulosa qilib aytadigan bo‘lsak, O‘zbekiston yer yuzidagi ko‘plab mamlakatlar bilan turli sohalarda o‘zaro manfaatli aloqalarni keng yo‘lga qo‘yib kelmoqda, xalqaro xavfsizlik va barqarorlikni mustahkamlash, hamkorlikning barcha shakllarini rivojlantirish yo‘lida izchillik va qat'iyat bilan odimlamoqda. Jahon miqyosida raqobat, qarama-qarshilik, turli manfaatlar to‘qnashuvi, geopolitik ziddiyatlar tobora kuchayib borayatgan bu zamonda biz kuchli tashqi siyosat olib bormasdan turib, ko‘zlagan maqsadlarimizga erisha olmaymiz. REFERENCES 1. O‘zbekiston Respublikasi Prezidentining rasmiy veb-sayti. https://president.uz/uz/1422 2. O‘zbekiston Respublikasi Qonun hujjatlari ma'lumotlari milliy bazasi. https://lex.uz/ru/docs/- 452255?ONDATE=25.05.1992%2000#-452450 REFERENCES 1. O‘zbekiston Respublikasi Prezidentining rasmiy veb-sayti. https://president.uz/uz/1422 2. O‘zbekiston Respublikasi Qonun hujjatlari ma'lumotlari milliy bazasi. https://lex.uz/ru/docs/- 452255?ONDATE=25.05.1992%2000#-452450 3. O‘zbekiston Liberal-demokratik partiyasining rasmiy veb-sayti. https://uzlidep.uz/uz/news-of-uzbekistan/7998 4. O‘zbekiston Respublikasi Tashqi ishlar vazirligi qoshidagi Diplomatik korpusga xizmat ko‘rsatish byurosining rasmiy veb-sayti. http://www.ddsmfa.uz/uz/ozbekiston-prezidenti-shavkat-mirziyoyevning- tashqiishlar-vazirligi-va-mamlakatimizning-xorijiy 4. O‘zbekiston Respublikasi Tashqi ishlar vazirligi qoshidagi Diplomatik korpusga xizmat ko‘rsatish byurosining rasmiy veb-sayti. http://www.ddsmfa.uz/uz/ozbekiston-prezidenti-shavkat-mirziyoyevning- tashqiishlar-vazirligi-va-mamlakatimizning-xorijiy 5. O‘zbekiston tarixi, o‘rta ta'lim maktablarining 11-sinf o‘quvchilari va o‘rta maxsus, kasb-hunar ta'lim muassasalari uchun darslik – T.: G‘afur G‘ulom, 2018 5. O‘zbekiston tarixi, o‘rta ta'lim maktablarining 11-sinf o‘quvchilari va o‘rta maxsus, kasb-hunar ta'lim muassasalari uchun darslik – T.: G‘afur G‘ulom, 2018 Volume-15, Issue-1, August- 2022 www.pedagoglar.uz 22
https://openalex.org/W3044616152	https://escholarship.org/content/qt1nt70759/qt1nt70759.pdf?t=qhd6bi	English	null	Novice Physician Ultrasound Evaluation of Pediatric Tricuspid Regurgitant Jet Velocity	The western journal of emergency medicine/Western journal of emergency medicine	2,020	cc-by	5,338	UC Irvine Western Journal of Emergency Medicine: Integrating Emergen Care with Population Health Title Novice Physician Ultrasound Evaluation of Pediatric Tricuspid Regurgitant Jet Velocity Permalink https://escholarship.org/uc/item/1nt70759 Journal Western Journal of Emergency Medicine: Integrating Emergency Care with Population Health, 21(4) ISSN 1936-900X Authors Binder, Zachary W. O'Brien, Sharon E. Boyle, Tehnaz P. et al. Publication Date 2020 DOI 10.5811/westjem.2020.3.45882 Copyright Information Copyright 2020 by the author(s).This work is made available under the terms of a Creat Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine Western Journal of Emergency Medicine: Integrating Emergen Care with Population Health Title Novice Physician Ultrasound Evaluation of Pediatric Tricuspid Regurgitant Jet Velocity Permalink https://escholarship.org/uc/item/1nt70759 Journal Western Journal of Emergency Medicine: Integrating Emergency Care with Population Health, 21(4) ISSN 1936-900X Authors Binder, Zachary W. O'Brien, Sharon E. Boyle, Tehnaz P. et al. Publication Date 2020 DOI 10.5811/westjem.2020.3.45882 Copyright Information Copyright 2020 by the author(s).This work is made available under the terms of a Creat Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine Copyright Information Copyright 2020 by the author(s).This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Novice Physician Ultrasound Evaluation of Pediatric Tricuspid Regurgitant Jet Velocity Zachary W. Binder, MD* Sharon E. O’Brien, MD* Tehnaz P. Boyle, MD, PhD* Howard J. Cabral, PhD† Sepehr Sekhavat, MD* Joseph R. Pare, MD, MHS‡ Boston Medical Center, Boston University School of Medicine, Department of Pediatrics, Boston, Massachusetts Boston University School of Public Health, Department of Biostatistics, Boston, Massachusetts Boston Medical Center, Boston University School of Medicine, Department of Emergency Medicine, Boston, Massachusetts * † ‡ Introduction: Pulmonary hypertension, associated with high mortality in pediatric patients, is traditionally screened for by trained professionals by measuring a tricuspid regurgitant jet velocity (TRJV). Our objective was to test the feasibility of novice physician sonographers (NPS) to perform echocardiograms of adequate quality to exclude pathology (defined as TRJV > 2.5 meters per second Methods: We conducted a cross-sectional study of NPS to assess TRJV by echocardiogram in an urban pediatric emergency department. NPS completed an educational course consisting of a didacti curriculum and hands-on workshop. NPS enrolled a convenience sample of patients aged 7-21 years. Our primary outcome was the proportion of echocardiograms with images of adequate quality to exclude pathology. Our secondary outcome was NPS performance on four image elements. We present descriptive statistics, binomial proportions, kappa coefficients, and logistic regression analysis Results: Eight NPS completed 80 echocardiograms. We found 82.5% (95% confidence interval [CI], 74.2-90.8) of echocardiograms had images of adequate quality to exclude pathology. Among image elements, NPS obtained a satisfactory, apical 4-chamber view in 85% (95% CI, 77.1-92.9); positioned the color box accurately 65% (95% CI, 54.5-75.5); optimized TRJV color signal 78.7% (95% CI, 69.8- 87.7); and optimized continuous-wave Doppler in 55% (95% CI, 44.1-66.0) of echocardiograms. Conclusion: NPS obtained images of adequate quality to exclude pathology in a majority of studie however, optimized acquisition of specific image elements varied. This work establishes the basis for future study of NPS assessment of TRJV pathology when elevated pulmonary pressures are of clinical concern. [West J Emerg Med. 2020;21(4)1029–1035.] Introduction: Pulmonary hypertension, associated with high mortality in pediatric patients, is traditionally screened for by trained professionals by measuring a tricuspid regurgitant jet velocity (TRJV). Our objective was to test the feasibility of novice physician sonographers (NPS) to perform echocardiograms of adequate quality to exclude pathology (defined as TRJV > 2.5 meters per second). Methods: We conducted a cross-sectional study of NPS to assess TRJV by echocardiogram in an urban pediatric emergency department. Powered by the California Digital Library University of California eScholarship.org eScholarship.org Original Research Novice Physician Ultrasound Evaluation of Pediatric Tricuspid Regurgitant Jet Velocity NPS completed an educational course consisting of a didactic curriculum and hands-on workshop. NPS enrolled a convenience sample of patients aged 7-21 years. Our primary outcome was the proportion of echocardiograms with images of adequate quality to exclude pathology. Our secondary outcome was NPS performance on four image elements. We present descriptive statistics, binomial proportions, kappa coefficients, and logistic regression analysis. Results: Eight NPS completed 80 echocardiograms. We found 82.5% (95% confidence interval [CI], 74.2-90.8) of echocardiograms had images of adequate quality to exclude pathology. Among image elements, NPS obtained a satisfactory, apical 4-chamber view in 85% (95% CI, 77.1-92.9); positioned the color box accurately 65% (95% CI, 54.5-75.5); optimized TRJV color signal 78.7% (95% CI, 69.8- 87.7); and optimized continuous-wave Doppler in 55% (95% CI, 44.1-66.0) of echocardiograms. Conclusion: NPS obtained images of adequate quality to exclude pathology in a majority of studies; however, optimized acquisition of specific image elements varied. This work establishes the basis for future study of NPS assessment of TRJV pathology when elevated pulmonary pressures are of clinical concern. [West J Emerg Med. 2020;21(4)1029–1035.] hypertension first present with syncope.1 The gold standard for diagnosing pulmonary hypertension is cardiac catheterization.4 Alternatively, cardiologists estimate pulmonary artery pressure by measuring a tricuspid regurgitant jet velocity (TRJV) during transthoracic echocardiography. An elevated TRJV of > 2.5 meters per second (m/s) is a surrogate measure of elevated pulmonary artery pressure in lieu of invasive cardiac catheterization (TRJV pathology).5,6 While a pathologic Background Pulmonary hypertension, while rare in children and young adults, carries a 25% five-year mortality.1 Pulmonary hypertension is a clinical consideration during various pediatric emergency department (PED) presentations including syncope and pulmonary embolism.2,3 Thirty-six percent of children with idiopathic or familial pulmonary Western Journal of Emergency Medicine Volume 21, no. 4: July 2020 1029 Binder et al. Novice Physician Ultrasound Evaluation of Pediatric TRJV Population Health Research Capsule What do we already know about this issue? Emergency physician sonographers have previously been shown to accurately assess many components of a point-of-care echocardiogram. What was the research question? Are novice physician sonographers able to perform echos that exclude tricuspid regurgitant jet (TRJ) pathology What was the major finding of the study? Pediatric cardiologist rated 82.5% of echos performed to be of adequate quality to exclude TRJ pathology. How does this improve population health? This pilot study establishes the basis for future investigation of novice assessment of TRJ pathology when elevated pulmonary pressures are of clinical concern. TRJV is rare in children and young adults, approximately 68-86% of healthy individuals will have a non-pathologic TRJV (detectable jet velocity up to 2.5 m/s).7-9 The remaining 14-32% of healthy children and young adults will have no tricuspid regurgitant jet. Population Health Research Capsule What do we already know about this issue? Emergency physician sonographers have previously been shown to accurately assess many components of a point-of-care echocardiogram. p g g j To date, TRJV has only been reliably measured by cardiologists and ultrasound technicians with cardiologist oversight. Unfortunately, this level of expertise, which is the current standard of care, is often unavailable to emergency physicians at times when patients may have concerning clinical presentations.10 Emergency physician sonographers have previously been shown to accurately assess and measure many components of a point-of-care echocardiogram.11-16 Point-of-care ultrasound, in comparison to comprehensive ultrasound, is an abbreviated examination performed by a medical professional at the bedside, which is used to identify either the presence or absence of specific pathologic findings. Previous studies have used a combination of didactic instruction and practical training to teach specific components of a point-of-care echocardiogram to emergency physicians.11-16 To our knowledge, the ability of novice physician sonographers (NPS) to perform assessments of TRJV has not been previously studied. As a result, we performed this pilot study aimed at determining feasibility. What was the research question? Goals of Investigation prior to enrollment. All patients less than 18 years of age provided verbal assent. The Boston University Medical Campus and Boston Medical Center Institutional Review Board approved this study. Our study objectives were to 1) test the feasibility of NPS to perform echocardiograms in the PED of adequate quality to exclude pathology (defined as TRJV > 2.5 m/s) as determined by a blinded pediatric cardiologist, and 2) identify patient and NPS characteristics associated with adequately performed echocardiograms. Novice Physician Sonographer Population We aimed to analyze the performance of NPS, which we defined as individuals who had performed fewer than 50 lifetime echocardiograms. This number was selected based on the 2016 ACEP policy statement.17 The primary investigator (PI) recruited EM residents, pediatric EM fellows, and pediatric EM attendings as unpaid volunteers to participate as NPS based on their limited echocardiography experience. All of the institution’s first-year EM residents (14), eligible pediatric EM fellows (2), and eligible pediatric EM attendings (5) were first contacted by email, followed by in-person recruitment if interest in participation was expressed. METHODS Study Design We conducted a prospective cross-sectional study from April 2018 to October 2018 in an urban, tertiary care facility with an annual census of 24,000 PED visits. The facility is a Level I trauma center with an accredited pediatric emergency medicine (PEM) fellowship and EM residency. The facility operates a separate PED that cares for patients through 21 years of age. Background Are novice physician sonographers able to perform echos that exclude tricuspid regurgitant jet (TRJ) pathology What was the major finding of the study? Pediatric cardiologist rated 82.5% of echos performed to be of adequate quality to exclude TRJ pathology. How does this improve population health? This pilot study establishes the basis for future investigation of novice assessment of TRJ pathology when elevated pulmonary pressures are of clinical concern. Western Journal of Emergency Medicine Educational Intervention We obtained consensus on all proposed modifications using modified Delphi methodology.21 The final curriculum contained four elements: 1) apical 4-chamber view; 2) color box positioning; 3) TRJV color signal optimization; and 4) continuous-wave Doppler interrogation (Figure 1 and Figure 2). The study’s PI and primary pediatric cardiologist (SO) administered the didactic curriculum. D Immediately following the didactic curriculum, NPS participated in a hands-on workshop consisting of deliberate practice with direct feedback from experts (professional cardiac sonographer and certified pediatric cardiologist). All NPS were required to complete a proctored echocardiogram during the workshop where an expert assessed performance. The proctored examination required the successful completion of all four image elements. Proctored echocardiograms established the novice’s ability to obtain TRJV images prior to his or her participation in the study. Figure 2. Tricuspid regurgitant jet velocity image elements. 2A. Apical 4-Chamber, 2B. Color box positioning, 2C. Tricuspid regurgitant jet signal optimization, 2D. Continuous-wave Doppler interrogation. Apical 4-Chamber View 1. Image orientation with cardiac apex at the top of screen and left ventricle to the right of screen Sample Size Estimation We predicted 80% of echocardiograms would have images of adequate quality to exclude pathology (TRJV > 2.5 m/s). Based on this estimate, we required 80 echocardiograms to generate a 95% confidence interval (CI) with a lower bound percentage of 72.6%. Therefore, each of the eight NPS was asked to perform a minimum of 10 echocardiograms over a six-month period. 6. Color box width minimized to just include tricuspid valve orifice 7. Clip saved showing a dynamic sweep through the tricuspid valve (anterior posterior) or (posterior anterior) 8. Select probe position that generates maximal regurgitant color signal Image Rating Doppler cursor aligned parallel to color jet flow 11. Doppler gain adjusted to maximize waveform 12. Baseline adjusted to maximize display of wave form 13. Image includes three full cardiac cycles 14. Image saved with continuous-wave Doppler applied Educational Intervention NPS screened and enrolled a convenience sample of patients aged 7-21 years who presented to the PED. Medical records were reviewed prior to approaching subjects for enrollment. We excluded patients who were critically ill, non- English speaking, or were younger than seven years of age. Patients less than seven years of age were excluded at the recommendation of the study’s primary cardiologist to limit exclusions based on patient intolerance. All other patients were eligible for enrollment. The patient or patient’s guardian (for minors under 18 years of age) provided informed consent Prior to participation in the study, NPS completed a three-hour educational course combining a didactic curriculum (30 minutes) and hands-on workshop (2.5 hours). The educational course taught NPS the steps required to obtain TRJV images. The didactic curriculum used still- image and video-clip modalities to demonstrate a stepwise approach to obtaining a TRJV. As no curriculum for TRJV image acquisition exists, the study’s principal investigator (PI) and primary pediatric cardiologist (SO) created a Volume 21, no. 4: July 2020 Western Journal of Emergency Medicine 1030 Novice Physician Ultrasound Evaluation of Pediatric TRJV Binder et al. Figure 2. Tricuspid regurgitant jet velocity image elements. 2A. Apical 4-Chamber, 2B. Color box positioning, 2C. Tricuspid regurgitant jet signal optimization, 2D. Continuous-wave Doppler interrogation. A B C D curriculum through a comprehensive multistep process. A literature review of pediatric echocardiography reference materials led to the generation of an initial curriculum.18-20 A panel of three independent pediatric cardiologists from outside the study institution reviewed the curriculum and provided feedback. These cardiologists had no role in curriculum generation, or image rating. We obtained consensus on all proposed modifications using modified Delphi methodology.21 The final curriculum contained four elements: 1) apical 4-chamber view; 2) color box positioning; 3) TRJV color signal optimization; and 4) continuous-wave Doppler interrogation (Figure 1 and Figure 2). The study’s PI and primary pediatric cardiologist (SO) administered the didactic curriculum. curriculum through a comprehensive multistep process. A literature review of pediatric echocardiography reference materials led to the generation of an initial curriculum.18-20 A panel of three independent pediatric cardiologists from outside the study institution reviewed the curriculum and provided feedback. These cardiologists had no role in curriculum generation, or image rating. Image Rating The study’s primary cardiologist, SO, blinded to NPS and patient identity, reviewed each recorded study. The cardiologist first reviewed all images to determine whether they could confidently exclude pathology based on the images provide. The cardiologist then performed a secondary analysis where individual image elements were assessed (Figure 1). The cardiologist recorded these results on an electronic score sheet. To assess inter-rater reliability a second pediatric cardiologist, not otherwise involved in the study, independently reviewed 20% of all study echocardiograms. Figure 1. Tricuspid regurgitant jet velocity curriculum. Apical 4-Chamber View 1. Image orientation with cardiac apex at the top of screen and left ventricle to the right of screen 2. Outline of all four chambers simultaneously visualized 3. Image aligned with ultrasound beam parallel to intraventricular septum and perpendicular to tricuspid valve 4. Image saved of apical 4-chamber view Color Box Positioning 5. Color box extending from the back wall of right atrium past the tricuspid valve leaflet tips 6. Color box width minimized to just include tricuspid valve orifice Tricuspid Regurgitant Jet Velocity Color Signal Optimization 7. Clip saved showing a dynamic sweep through the tricuspid valve (anterior posterior) or (posterior anterior) 8. Select probe position that generates maximal regurgitant color signal Continuous-wave Doppler 9. Doppler cursor placed in the middle of tricuspid regurgitant color jet 10. Doppler cursor aligned parallel to color jet flow 11. Doppler gain adjusted to maximize waveform 12. Baseline adjusted to maximize display of wave form 13. Image includes three full cardiac cycles 14. Image saved with continuous-wave Doppler applied Figure 1. Tricuspid regurgitant jet velocity curriculum. Apical 4-Chamber View 1. Image orientation with cardiac apex at the top of screen and left ventricle to the right of screen 2. Outline of all four chambers simultaneously visualized 3. Image aligned with ultrasound beam parallel to intraventricular septum and perpendicular to tricuspid valve 4. Image saved of apical 4-chamber view Color Box Positioning 5. Color box extending from the back wall of right atrium past the tricuspid valve leaflet tips 6. Color box width minimized to just include tricuspid valve orifice Tricuspid Regurgitant Jet Velocity Color Signal Optimization 7. Clip saved showing a dynamic sweep through the tricuspid valve (anterior posterior) or (posterior anterior) 8. Select probe position that generates maximal regurgitant color signal Continuous-wave Doppler 9. Doppler cursor placed in the middle of tricuspid regurgitant color jet 10. Data Collection NPS recorded still images and video clips in a protocolized fashion using a Philips SPARQ (Philips Healthcare, Bothell, WA) ultrasound machine. Images automatically transferred wirelessly to Qpath (Telexy Healthcare, Maple Ridge, BC) software, a program for storage and management of ultrasound examinations. NPS obtained all images and clips using a standardized imaging preset with a phased array probe (S4-2). We de-identified all study images at the time of acquisition with a study identification. 9. Doppler cursor placed in the middle of tricuspid regurgitant color jet 10. Doppler cursor aligned parallel to color jet flow 11. Doppler gain adjusted to maximize waveform 12. Baseline adjusted to maximize display of wave form 13. Image includes three full cardiac cycles Figure 1. Tricuspid regurgitant jet velocity curriculum. 14. Image saved with continuous-wave Doppler applied Volume 21, no. 4: July 2020 Western Journal of Emergency Medicine 1031 Binder et al. Novice Physician Ultrasound Evaluation of Pediatric TRJV Table 1. Characteristics of patients and novice physician sonographers (NPS) in study to determine whether NPS could obtain point-of-care echocardiogram images of adequate quality to exclude pathology. NPS completed a standardized data collection form after each echocardiogram. The study’s PI abstracted patient information including gender, age, ethnicity, vital signs, and body mass index (BMI) from the electronic health record. The study’s PI transcribed clinical data into Research Electronic Data Capture (REDCap) (Nashville, TN).22 Patient characteristics (n = 73) Median [IQR] Age (years) 19 [17-20] BMI (kg/m²)* 23.9 [21.9-27.5] Gender (female) 40/73 (54.8%; 95% CI, 43.3-66.3) Ethnicity (Hispanic) 19/73 (26.0%; 95% CI, 15.9-36.1) Patient vital signs (n = 73) Median [IQR] Temperature (°F) 99.0 [97.3-98.7] Heart rate (beats/minute) 79 [68-88] Respiratory rate (breaths/minute) 18 [16-18] Systolic blood pressure (mmHg) 120 [112-124] Diastolic blood pressure (mmHg) 74 [68-78] Oxygen saturations (%) 99 [98-100] Novice physician sonographer level of training (n = 8) n (%) Emergency medicine residents 2 (25.0%) Pediatric emergency medicine fellows 2 (25.0%) Pediatric emergency medicine attendings 4 (50.0%) N = 52 for this variable. IQR, interquartile range; kg/m2, kilograms per meter squared; °F, degrees Fahrenheit; mmHg, millimeters of mercury; BMI, body mass index; CI, confidence interval. Demographics NPS consented 75 eligible patients for participation during the six-month study period. Two consented patients did not have echocardiograms performed because of time constraints. Ultimately 73 patients, ages 7-21 years, provided 80 echocardiograms for analysis (seven patients provided two echocardiograms by different NPS). Table 1 details patient and NPS characteristics. DISCUSSION The study found 82.5% of echocardiograms to have images of adequate quality to exclude pathology, TRJV >2.5 m/s. We believe this provides preliminary evidence NPS can perform adequate TRJV studies following a brief educational intervention. This is in line with prior studies showing novices can accurately assess and measure other focused components of a point-of-care echocardiogram.11-16 Primary Outcome The study’s primary pediatric cardiologist rated 66 of 80 (82.5%, 95% CI, 74.2-90.8) echocardiograms to have images of adequate quality to exclude pathology. The remaining 14 of 80 were deemed to be of too poor quality to assess for the presence of TRJV. Of 66 echocardiograms, 27 (40.9%) had no TRJ present, 21 (31.8%) had a present but not measurable TRJ, and 18 (27.3%) had a measurable TRJV less than or equal to 2.5 m/s. None of the echocardiograms had a TRJV greater than 2.5 m/s (Figure 3). Western Journal of Emergency Medicine Statistical Analysis Descriptive statistics, binomial proportions, and kappa coefficients were performed to analyze the data. We tested for patient and NPS characteristics associated with adequately performed echocardiograms using logistic regression analysis. All statistical analyses were performed using SAS 9.4 software (SAS Institute, Cary, NC). Secondary Outcomes Of the four image elements, the proportion of satisfactorily completed elements ranged from 55% (95% CI, 44.1- 66.0) for the interrogation of continuous-wave Doppler to 85% (95% CI, 77.1- 92.9) for the acquisition of apical 4-chamber view (Table 2A). To complete the examinations, NPS took an average of 2.5 minutes (interquartile range [IQR] 1.6 - 4.7) from time of first to last saved ultrasound image timestamp. NPS performed an average of 11 echocardiograms (IQR 8-13) (Table 2B). It is important to note for this study it was possible to obtain images of adequate quality to exclude pathology without performing each image element optimally. For example, NPS may have failed to adjust the display to produce a textbook image; however, the images may still have been adequate to exclude pathology when reviewed by the cardiologist. The objective of our secondary analysis was to determine which image elements were more difficult for NPS. They were most successful in acquiring an apical 4-chamber view (85%), and least successful in optimally interrogating continuous-wave Doppler (55%). The interrogation of continuous-wave Doppler is an advanced skill and this study likely represented the NPS’s first exposure to this function. These results provide further support that For the variables selected to test association with image quality, younger patient age was associated with improved echocardiogram adequacy (odds ratio [OR] 0.64; 95% CI, 0.41- 0.99; p=0.04). No association was found between patient gender or NPS level of clinical training and adequacy of images (Table 2C). There was fair agreement (κ = 0.25) between the two pediatric cardiologists when assessing the primary outcome, the ability to exclude pathology. Volume 21, no. 4: July 2020 1032 Binder et al. Novice Physician Ultrasound Evaluation of Pediatric TRJV 75 Patients consented 73 Patients scanned 73 Patients scanned 80 Echocardiograms 66 Adequate images 27 No TRJ present 21 TRJ present, not measurable 18 TRJ ≤ 2.5 m/s 0 TRJ > 2.5 m/s 14 Images of too poor quality 2 No scan (time constraints) Figure 3. Flowchart demonstrating patient enrollment and image rating as determined by pediatric cardiologist. Seven patients were scanned by two sonographers. TRJ, tricuspid regurgitant jet; m/s, meters per second. 75 Patients consented 73 Patients scanned 73 Patients scanned 80 Echocardiograms 14 Images of too poor quality 18 TRJ ≤ 2.5 m/s 21 TRJ present, not measurable Figure 3. Flowchart demonstrating patient enrollment and image rating as determined by pediatric cardiologist. Secondary Outcomes Seven patients were scanned by two sonographers. TRJ, tricuspid regurgitant jet; m/s, meters per second. NPS interpretation and incorporation of their echocardiograms into clinical care is needed. ultrasound performance is dependent on repeated exposure and practice.17,23,24 i We found a statistically significant association between younger patient age and echocardiograms with images of adequate quality. This association may be explained by thinner chest walls in younger patients; however, the study was inadequately powered to evaluate this relationship. Furthermore, the study was inadequately powered to determine a relationship between patient BMI and image adequacy. Western Journal of Emergency Medicine LIMITATIONS As a convenience sample of non-critically ill patients from a single center, this may limit the study’s generalizability. Our findings will require external validation in a future larger study prior to clinical implementation. While no patients in the study had a TRJV greater than 2.5 m/s, this was not unexpected. NPS successfully detected TRJVs at lower velocities. Further study would be required to demonstrate a NPS’s ability to rule in pathology. We feel that our patient population is representative of a general population because prior studies of healthy children and young adults found a similar distribution of TRJV findings.7-9 We found a fair level of inter-rater agreement. Liem et al demonstrated an inter-rater kappa of 0.45 (moderate agreement) when measuring TRJV.25 Their study analyzed measurements obtained by expert sonographers that were then interpreted by cardiologists. It is possible that images obtained by novices have a greater range of quality leading to a lower inter-rater agreement. Both our study and that by Liem et al suggest expert agreement regarding the assessment of TRJV is varied. The primary study outcome, adequacy of images, is subjective and therefore subject to bias. We found no objective assessment tool available for the purposes of our study. In an attempt to limit bias, we blinded the raters to patient and NPS information. In prior studies on this subject, experts have only been able to generate moderate inter-rater agreement suggesting the lack of an ideal assessment mechanism. It is notable that 82.5% of echocardiograms in our study had images of adequate quality to exclude pathology. As an initial pilot study, the first step was to determine ability regarding image acquisition. We recognize that future study of Volume 21, no. 4: July 2020 1033 Western Journal of Emergency Medicine Binder et al. Novice Physician Ultrasound Evaluation of Pediatric TRJV Novice Physician Ultrasound Evaluation of Pediatric TRJV 2A. Rating of TRJV image elements Ratio of satisfactory completion 95% CI Apical 4-chamber 68/80 (85.0%) (77.1-92.9) Color box positioning 52/80 (65.0%) (54.5-75.5) TRJ signal optimization 63/80 (78.7%) (69.8-87.7) Continuous-wave doppler 44/80 (55.0%) (44.1-66.0) 2B. Echocardiogram characteristics Median [IQR] Scans per sonographer 11 [8-13] Time to complete ultrasound study (minutes) 2.5 [1.6-4.7] 2C. Association with adequate echocardiogram OR [95% CI] P-value Patient age 0.64 [0.41-0.99] 0.04 Patient gender 0.58 [0.16-2.13] 0.41 Sonographer: resident vs attending 0.75 [0.15-3.78] 0.72 Sonographer: fellow vs attending 0.84 [0.17-4.18] 0.83 Table 2. Echocardiography results. *Logistic regression analysis. LIMITATIONS CI, confidence interval; TRJV, tricuspid regurgitant jet velocity; IQR, interquartile range; OR, odds ratio. quality of subspecialty input and referrals at the time of ED presentations. We acknowledge these evaluations are best done in consultation with cardiology colleagues and not as a replacement for their expertise. This study suggests that NPS can obtain images of adequate quality to evaluate TRJV in the absence of pathology (TRJV > 2.5 m/s) after a brief educational intervention. This work establishes the basis for future study of novice assessment of TRJV pathology when elevated pulmonary pressures are of clinical concern. The study’s primary cardiologist (SO) helped develop the initial curriculum and served as one of the study’s image raters. Three independent cardiologists not involved in the study, via a modified Delphi approach, finalized this curriculum. It is possible that by having the primary cardiologist assist with the initial curriculum development and grading the images bias was introduced into this study. We believe this would be limited by having the external pediatric cardiologists provide consensus on approving the final curriculum. Additionally, the two cardiologists were blinded to both patient and NPS identity when rating images. The study’s protocol did not incorporate pulsed-wave Doppler prior to continuous-wave Doppler analysis. This is an accepted practice in echocardiography and has been used in prior studies on the topic.26 The risk in excluding pulsed-wave Doppler is overestimating the TRJV through contamination of signal from extremely rare intracardiac shunting lesions. Identification of shunting lesions, while outside the scope of this study, represents pathology that one would ideally not miss. We do not believe the decision had an impact on our study’s results. Address for Correspondence: Zachary W. Binder, MD, Department of Pediatrics, Division of Pediatric Emergency Medicine, 55 Lake Avenue, North, Worcester, MA 01655. Email: Zachary.Binder@ bmc.org. Zachary.Binder@umassmemorial.org. Conflicts of Interest: By the WestJEM article submission agreement, all authors are required to disclose all affiliations, funding sources and financial or management relationships that could be perceived as potential sources of bias. No author has professional or financial relationships with any companies that are relevant to this study. There are no conflicts of interest or sources of funding to declare. CONCLUSION There is currently no way to easily assess pulmonary hypertension in the PED setting. The ability to assess for pulmonary hypertension in the PED could assist in the management of multiple patient presentations including syncope and pulmonary embolism. While this study was performed in a PED, we believe the results and potential clinical implications would also apply to an adult population. It is important to note that in the era of the electronic health record, if an emergency physician could perform a study and review the images with a cardiologist it might improve the Copyright: © 2020 Binder et al. This is an open access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) License. See: http://creativecommons.org/ licenses/by/4.0/ Western Journal of Emergency Medicine REFERENCES 1. Barst RJ, McGoon MD, Elliott CG, et al. Survival in childhood pulmonary arterial hypertension: Insights from the registry to evaluate Western Journal of Emergency Medicine Volume 21, no. 4: July 2020 1034 Binder et al. Novice Physician Ultrasound Evaluation of Pediatric TRJV early and long-term pulmonary arterial hypertension disease management. Circulation. 2012;125(1):113-22. 14. 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Executive summary of the American Heart Association and American Thoracic Society joint guidelines for pediatric pulmonary hypertension. Am J Respir Crit Care Med. 2016;194(7):898-906. 17. Tayal VS, Raio CC. ACEP Policy Statement - Ultrasound Guidelines: Emergency, Point-of-Care, and Clinical Ultrasound Guidelines in Medicine. 2016. Available at: https://www.acep.org/ globalassets/new-pdfs/policy-statements/ultrasound-guidelines- --emergency-point-of-care-and-clinical-ultrasound-guidelines-in- medicine.pdf. Accessed December 19, 2018. 5. Lanzarini L, Fontana A, Lucca E, et al. Noninvasive estimation of both systolic and diastolic pulmonary artery pressure from Doppler analysis of tricuspid regurgitant velocity spectrum in patients with chronic heart failure. Am Heart J. 2002;144(6):1087-94. 6. Bossone E, Bodini BD, Mazza A, et al. Pulmonary arterial hypertension : the key role of echocardiography pulmonary arterial hypertension. The key role of echocardiography. 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Educ Psychol. 1997;17:373-86. 9. Yoshida K, Yoshikawa J, Shakudo M, et al. Color Doppler evaluation of valvular regurgitation in normal subjects. Circulation. 1988;78(4):840-7. 22. Harris PA, Taylor R, Thielke R, et al. Research electronic data capture (REDCap): a metadata-driven methodology and workflow process for providing translational research informatics support. J Biomed Inform. 2009;42(2):377-81. 10. Moore CL, Lin H. Ultrasonography in community emergency departments in the United States : access to ultrasonography performed by consultants and status of emergency physician – performed ultrasonography. Ann Emerg Med. 2006;47(2):147-53. 23. Blehar DJ, Barton B, Gaspari RJ. Learning curves in emergency ultrasound education. Acad Emerg Med. 2015;22(5):574-82. 24. Gaspari RJ, Dickman E, Blehar D. Learning curve of bedside ultrasound of the gallbladder. J Emerg Med. 2009;37(1):51-6. 11. Mandavia DP, Hoffner RJ, Mahaney K, et al. Bedside echocardiography by emergency physicians. 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https://openalex.org/W3137633260	https://www.researchsquare.com/article/rs-194775/latest.pdf	English	null	NiO Nanoparticles: A Highly Efficient Catalyst For One-Pot Three-Component Synthesis of Pyrano[2, 3-d]Pyrimidine Derivatives in Green Media	Research Square (Research Square)	2,021	cc-by	3,535	NiO Nanoparticles: A Highly Efficient Catalyst For One-Pot Three-Component Synthesis of Pyrano[2, 3- d]Pyrimidine Derivatives in Green Media Elahe Sabbaghnasab Islamic Azad University Kerman Branch Enayatollah Sheikhhosseini (  sheikhhosseiny@gmail.com ) Islamic Azad University Kerman Branch https://orcid.org/0000-0003-2973-9768 Elahe Sabbaghnasab Islamic Azad University Kerman Branch Original Research Keywords: NiO nanoparticles, Pyrano [2, 3-d] pyrimidines, Barbituric acid, Multicomponent reaction Posted Date: February 15th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-194775/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/11 Page 1/11 Abstract In this paper NiO nanoparticles are utilized for effective strengthening of annulated pyrano [2, 3- d] pyrimidine synthesis through primary Knoevenagel, the following Micheal and ultimate heterocyclization reactions of aldehyde, malononitrile and barbituric acid. The NiO nanoparticles characteristics are identified through using advanced techniques like IR, UV, EDX, XRD, SEM and TEM. The nano-NiO particles are mostly of below < 100 nm sizes with uniform sphere shapes. The adopted approach is advantages due to its simple processing, relatively short reaction time, often good to high average yields, convenient workability, and environmental friendliness. 2.2 Material characterization The characterization of supplied materials was performed in terms of physical constants against authentic samples and FT-IR spectroscopy. Thin layer chromatography (TLC) on Merck pre-coated silica gel 60 F254 aluminum sheets as well as UV light visualization technique was used to monitor the products purity and the reactions’ progress. A BUCHI 510 melting point device was utilized for determining the material melting points. To record Fourier, transform infrared (FT-IR) spectroscopy a JASCO FT-IR-4000 spectrophotometer was used while the samples were dispersed on KBr pellets across 4000–400 cm−1 (Bruker, Germany) wavelength range. Also, a Bruker Advance DPX 400 MHz equipment was utilized for recording nuclear magnetic resonance (NMR) spectra, measured on DMSO-d6 relative to tetramethylsilane as an internal standard. Scanning of the X-ray diffraction (XRD) fresh patterns was performed in reflection mode through the use of a powder X‐ray diffractometer (X' Pert MPD, PAN analytical) equipped with Cu-Ka source of radiation (λ = 0.154 nm) between 10° and 90° (2‐Theta) having a stage width of 0.010″) using a Philips XPERT PRO (Bruker, model D8 Advance). The NiO nanoparticles morphology was examined via scanning electron microscopy (SEM) (Nano SEM 450) (FEI, U.S.). To perform Transmission electron microscope (TEM) measurements, a HITACHI H‐7650 electron microscope was used. To measure the UV–VIS spectra a USB-2000 UV–Vis spectrophotometer was used. 2.1 Materials All the chemical materials were supplied from Merck or Sigma Aldrich Co. and were used with no further treatment. We also used distilled water in this paper. Introduction The Nitrogen and Oxygen containing synthetic compounds like heterocyclic rings are very interesting because of possessing a wide spectrum of useful biological and pharmaceutical characteristic and activities [1-12]. Pyrano [2, 3-d] pyrimidines as an example, are structural motifs contained in synthetic and natural compounds that are biologically active. These heterocyclic rings exhibit invaluable biological healing activities, as they perform antitumor [13], hepatoprotective [14], antibronchitic [15] and anti-AIDS behaviours [16]. Accordingly, the efficient synthetization of such compounds and the relevant derivatives entail a motivating challenge for the organic chemistry researchers. Fortunately, Multicomponent reactions (MCRs) in modern organic synthetic chemistry are regarded highly applicable tools owing to having almost all necessary features to take part in ideal syntheses; features like high atom performance, fast and easy put up with highly time and energy-cost savings, environmentally friendliness as well as providing targeted and diversity-oriented syntheses [17]. Hence, currently devising new multicomponent reactions to produce biomedical and industrial synthetic scaffolds is indispensable. Numerous regular yet useful producers exist for producing the above compounds. From the above- mentioned techniques, the aromatic aldehydes, barbituric acid derivatives and malononitrile with their particular multi-component condensation reactions have been the focal of considerable focus and various bases have proved to be enduring catalysts for such reactions. Its noteworthy however that such protocols have their own drawbacks like corrosion, problematic catalyst recycling and waste generation issues [18-24]. Apparently, a potential of NPs application is using them as catalyst to develop new synthetic methods qualified for transformations in organic settings of environmentally friendly producers. According to some researchers, a number of nano-materials such as the metals metal oxides and NPs with semi-conductor properties could be utilized in organic chemistry as catalysts [25, 26]. Hence the development of new synthetic methods, facilitating the provision of organic compounds is most welcomed. Condensation reaction is among those chemical activities for which devising a new environmentally friendly catalyst could potentially provide an approach for dealing with the aforementioned challenges. Upon our previous study on synthesis of organic compounds by Page 2/11 nanoparticles [26-29], the scope of the present work was to achieve the multi-component pyrano [2, 3-d] pyrimidines by using NiO nano powders as a green, environmentally friendly catalyst (Scheme 1). nanoparticles [26-29], the scope of the present work was to achieve the multi-component pyrano [2, 3-d] pyrimidines by using NiO nano powders as a green, environmentally friendly catalyst (Scheme 1). 2.4 General preparation process of benzopyrano [2,3-d] pyrimidinones Aromatic aldehydes 1 (1.9 mmol), malononitrile 2 (2.2), barbituric acid or 2-thiobarbiturate 3 (2 mmol) and NiO nanoparticles (0.04 g) were taken in an RB flask with 10 mL H2O as solvent and stirred for appropriate time at reflux condition. Thin layer chromatography was used to monitor the reactions, after the completion of which 5 mL of DMSO was introduced into the reaction mixture. Afterwards the solid compound (catalyst) was separated through filtration, and water was added to the remaining solution so as to form precipitates. The precipitates were filtered and washed by ethanol to acquire the derivatives of pure product benzopyrano [2, 3-d] pyrimidine derivatives. Spectral data of the selected compound is given below: 7-Amino-2,3,4,5-tetrahydro-5-(3-nitrophenyl)-2,4-dioxo-1H-pyrano [2, 3-d] pyrimidine-6-carbonitrile (4a): M. P.: 260- 265 0C, IR (KBr, Cm-1): 1679, 2200, 3314. 1H NMR (400 MHZ, DMSO- d6) δ: 4.51 (s, 1H, CH), 7.32 (2H, brs- NH2), 7.61 (1H, t, J = 8.0H, H-Ar), 8.10- 8.11 (m, 2H, H- Ar), 11.72 (s, 1H, NH), 12.47 (s,1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 35.4, 58.3, 88.3, 119.6, 122.8, 122.9, 130.6, 135.3, 147.2, 148.5, 150.4, 153.2, 158.9, 163.2 ppm. 2.3 General process of the NiO nanoparticles synthesis Nickel oxide nanoparticles underwent synthetization in free standing powder form using wet chemical synthesis technique. In this study a dissolution of 1 mmol of Nickel (II) nitrate hexahydrate [Ni(NO3)2·6H2O, Merck] was prepared in 10 mL of polyethylene glycol [H(OCH2CH2)n OH), Merck]. A magnetic stirrer was then used to stir the solutions for 1 h at 60°C. Additionally, separate solutions of 2 mmol NaOH and 4 mmol sodium dodecyl sulfate were prepared through dissolution in 10 mL polyethylene glycol, each stirred for 1 h using a magnetic stirrer at 60°C; the prepared solution were then both added to Nickel (II) nitrate solution and stirred using magnetic stirrer for 2 h. at 60°C. The mixture so prepared underwent 450, 650 and 750 w irradiation for a 4 min time span then left to cool down to reach ambient temperature. Afterwards the centrifugation method was used to form the precipitates; they were then rinsed frequently by ethanol and dried in vacuum oven apparatus at 60°C to finally accumulate the nickel oxide nanoparticles. Page 3/11 Page 3/11 3.1 Characterization of nickel oxide nanoparticles To investigate the impacts of microwave on the nickel oxide nanoparticlesʼ synthesis, the nickel oxide nanoparticles were prepared by applying microwave various power levels (450, 650 and 750 W). Figs. (1a, 1b and 1c) respectively illustrate the NiO nanoparticles scanning electron microscope images. The SEM image represented a spherical structure of the NiO nanoparticles, revealing that they were distributed widely under the effect of agglomeration, probably due to the presence of sodium dodecyl sulfate. The below fig. (1a) SEM image of NiO nanoparticles demonstrates an area of narrow distribution of 50-60 nm particle size, suggesting that 450 w is providing the suitable radiation power condition. As can be seen from the obtained TEM images, the NiO nanoparticles are spherical in shape. The TEM image Fig. (2) shows that NiO mostly spherical nanoparticle sizes lie somewhere between 40-50 nm, while heavy agglomeration can be spotted in some areas. In this study the TEM and SEM imaging systems measurements confirmed the average particle size obtained from Debye-Scherer formula through XRD patterns. Infrared spectrometer (IR) measuring NiO nanoparticles exhibited some absorption bonds at 1409 cm- 1and 11590 cm-1 relative to NiO spinal structure. Also 600-1200 cm-1(Ni-O), 2850 and 2918 (CH2-SDS) and 1082, 1362 (S=O-SDS) adsorption bonds were consistent with the SDS-based synthesized NiO nanoparticles. Page 4/11 The following main elements were obtained from the energy dispersive X-ray (EDX) spectroscopy results: Ni (34%), O (28%), C (24%) and Na (18%). The NiO nanonoparticlesʼ XRD spectrum, synthesized under microwave optimal irradiations condition has been illustrated in Fig. (3). According to the Xpert high score software data, the synthesized sample XRD pattern with its JCPD card No. 47-1049 acknowledges the fruitful synthesis of NiO nanoparticles at 0.058 S-1 scanning rate across 10° to 80° in spinal phase spectrum. The below XRD pattern also contains –in addition to the NO diffusers–the Ni (OH)2 and SDS diffractions. A significant absorption peak of about 260 nm can be seen in the UV-VIS spectra of NiO nanoparticles. 3.2. Synthesis of pyrano [2, 3-d] pyrimidine derivatives by using NiO nanoparticles Page 5/11 Page 5/11 In summary, by employing the multi-component reaction of malononitrile, barbituric acid, and aromatic aldehydes, in the presence of nanoparticles of NiO as a catalyst, an efficient and rapid synthesis route was developed for pyrano [2,3-d] pyrimidine derivatives in this study resulting in good yields. One can cite several advantages for the same synthesis: it is simple to prepare NiO nanoparticle as the catalyst, the easy accessibility to the starting materials, high reaction times, the clean reaction profile, the easy workup and green media. In summary, by employing the multi-component reaction of malononitrile, barbituric acid, and aromatic aldehydes, in the presence of nanoparticles of NiO as a catalyst, an efficient and rapid synthesis route was developed for pyrano [2,3-d] pyrimidine derivatives in this study resulting in good yields. One can cite several advantages for the same synthesis: it is simple to prepare NiO nanoparticle as the catalyst, the easy accessibility to the starting materials, high reaction times, the clean reaction profile, the easy workup and green media. 3.2. Synthesis of pyrano [2, 3-d] pyrimidine derivatives by using NiO nanoparticles To extend the present study to experimental research, so that high performance MCRs are developed for the provision of annulated bioactive molecules of pyrimidine [29, 31], here we claim that in aqueous media, through one-pot three-component domino Knoevenagel- Michael addition reactions, the NiO nanoparticles led to catalysis of fast, efficient, and simple synthesis of pyrano[2,3-d]pyrimidine derivatives (Scheme 1). Initially, as a model reaction, the three-component reaction of 3-nitrobenzaldehyde 1a, barbituric acid 2a, and malononitrile 3 was employed to examine different solvents including: MeOH, EtOH, H2O, CHCl3 and CH2Cl2 (Table 1) in the presence of NiO nanoparticles (0.04 g). The appropriate chosen solvent for utilization in the reaction was H2O. Consequently, as Table 1 shows, the three-component model reaction was conducted while various amounts of catalysts were available. In order to come upon the optimal amount of catalyst for accessing pyrano [2, 3-d] pyrimidine 4a, the above investigation was conducted. According to the results, H2O with 0.04 g of NiO nanoparticles as the catalyst was the optimal reaction media for the formation of the products 4a. Once the reaction conditions were optimized, the suitability and effectiveness of the same procedure were surveyed by conducting the reaction through various readily accessible and simple substrates under the optimum conditions. Thus, under the optimum conditions, different aromatic aldehydes containing electron‐donating and electron‐withdrawing groups, namely, NO2, N, N-dimethyl and OCH3 in the para and ortho positions of the benzene ring were came into reaction with malononitrile and barbituric or thiobarbituric acid. As entries 1–12 in Table 4 show, within short reaction times, the concerned products were achieved in moderate-good isolated yields. Also, as entries 8-12 in Table 2 suggest, under the optimum conditions, thiobarbiturate acid was employed, giving high yields of the desired products. The proposed mechanism of the one-pot reaction between benzaldehyde derivatives, malononitrile and barbituric acid was described in Scheme 2. The acidic surface probably coordinates with the nitrogen and oxygen of the carbonyl carbon on which a partial positive charge appears. Based on this proposed mechanism, NiO nanoparticles are capable of activating aldehyde carbonyl group whilst accelerating barbituric acid enolization. The higher reactivity of the iminium group is utilized to facilitate Knoevenagel condensation between benzaldehyde 1 and malononitrile 2, which produce intermediate 6 that is attacked by the enolized barbituric acid 5 for generation of product 4 after proton transfer and tautomerization of intermediate 7 (Scheme 2). References 1. M. Ghashang, M. Kargar, M. R. Shafiee, S.S. Mansoor, A. Fazlinia, H. 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Ghazanfari, Microwave‐associate synthesis of Co3O4 nanoparticles as an effcient nanocatalyst for the synthesis of arylidene barbituric and Meldrum's acid derivatives in green media. Appl. Organomet. Chem., 33(9), e5100.G (2019). 30. M. Yahyazadehfar, E. Sheikhhosseini, S.A. Ahmadi, D. Ghazanfari, Microwave‐associate synthesis of Co3O4 nanoparticles as an effcient nanocatalyst for the synthesis of arylidene barbituric and Meldrum's acid derivatives in green media. Appl. Organomet. Chem., 33(9), e5100.G (2019). 31. E. Sheikhhosseini, D. Ghazanfari, V. Nezamabadi, 2013. A new method for synthesis of tetrahydrobenzo [b] pyrans and dihydropyrano [c] chromenes using p-dodecylbenzenesulfonic acid as catalyst in water. Iran. J. Catal., 3(4), 197-201 (2013). Tables Table. 1 Optimization of reaction condition for the yield of 7-amino-2,3,4,5-tetrahydro-5-(3- nitrophenyl)-2,4-dioxo-1H-pyrano[2,3-d]pyrimidine-6-carbonitrile (4a) Page 8/11 Table. 2 Pyrano[2,3-d]pyrimidine derivatives obtained by the reaction of aromatic aldehydes, malononitrile with barbituric and thiobarbituric acid using NiO nanoparticles as catalyst Table. 2 Pyrano[2,3-d]pyrimidine derivatives obtained by the reaction of aromatic aldehydes, malononitrile with barbituric and thiobarbituric acid using NiO nanoparticles as catalyst Figures Page 9/11 Page 9/11 Figure 3 XRD pattern of the NiO nanoparticle. Figure 1 a SEM image of the NiO nanoparticles (450 W), b SEM image of the NiO nanoparticle image of the NiO nanoparticles (750 W). Figure 2 a SEM image of the NiO nanoparticles (450 W), b SEM image of the NiO nanoparticles (650 W), c SEM image of the NiO nanoparticles (750 W). Figure 2 Figure 2 TEM image of NiO nanoparticles. Page 10/11 Page 10/11 Figure 3 Figure 3 Figure 3 Scheme01.png Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Scheme01.png Scheme02.png Page 11/11
https://openalex.org/W3116236139	https://www.researchsquare.com/article/rs-16448/v5.pdf?c=1631884710000	English	null	Quantification of arterial, venous, and cerebrospinal fluid flow dynamics by magnetic resonance imaging under simulated micro-gravity conditions: a prospective cohort study	Fluids and barriers of the CNS	2,021	cc-by	6,065	Quantification of Arterial, Venous, and Cerebrospinal Fluid Flow Dynamics by Magnetic Resonance Imaging Under Simulated Micro-Gravity Conditions: A Prospective Cohort Study Arslan Zahid (  arslanz1010@gmail.com ) Emory University School of Medicine https://orcid.org/0000-0002-4586-6751 Bryn Martin University of Idaho Stephanie Collins Georgia Institute of Technology John N. Oshinski Emory University Christopher Ross Ethier Georgia Institute of Technology Research Keywords: Spaceflight-Associated Neuro-Ocular Syndrome, head-down tilt, arterial, venous, cerebrospinal fluid dynamics, simulated microgravity Posted Date: January 13th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-16448/v5 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 on February 12th, 2021. See the published Quantification of Arterial, Venous, and Cerebrospinal Fluid Flow Dynamics by Magnetic Resonance Imaging Under Simulated Micro-Gravity Conditions: A Prospective Cohort Study Arslan Zahid (  arslanz1010@gmail.com ) Emory University School of Medicine https://orcid.org/0000-0002-4586-6751 Bryn Martin University of Idaho Stephanie Collins Georgia Institute of Technology John N. Oshinski Emory University Christopher Ross Ethier Georgia Institute of Technology Research Keywords: Spaceflight-Associated Neuro-Ocular Syndrome, head-down tilt, arterial, venous, cerebrospinal fluid dynamics, simulated microgravity Posted Date: January 13th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-16448/v5 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 on February 12th, 2021. See the published Research Version of Record: A version of this preprint was published on February 12th, 2021. See the published version at https://doi.org/10.1186/s12987-021-00238-3. Page 1/16 Abstract Background: Astronauts undergoing long-duration spaceflight are exposed to numerous health risks, including Spaceflight-Associated Neuro-Ocular Syndrome (SANS), a spectrum of ophthalmic changes that can result in permanent loss of visual acuity. The etiology of SANS is not well understood but is thought to involve changes in cerebrovascular flow dynamics in response to microgravity. There is a paucity of knowledge in this area; in particular, cerebrospinal fluid (CSF) flow dynamics have not been well characterized under microgravity conditions. Our study was designed to determine the effect of simulated microgravity (head-down tilt [HDT]) on cerebrovascular flow dynamics. We hypothesized that microgravity conditions simulated by acute HDT would result in increases in CSF pulsatile flow. Methods: In a prospective cohort study, we measured flow in major cerebral arteries, veins, and CSF spaces in fifteen healthy volunteers using phase contrast magnetic resonance (PCMR) before and during 15° HDT. Results: We found a decrease in all CSF flow variables [systolic peak flow (p=0.009), and peak-to-peak pulse amplitude (p=0.001)]. Cerebral arterial average flow (p=0.04), systolic peak flow (p=0.04), and peak- to-peak pulse amplitude (p=0.02) all also significantly decreased. We additionally found a decrease in average cerebral arterial flow (p=0.040). Finally, a significant increase in cerebral venous cross-sectional area under HDT (p=0.005) was also observed. Conclusions: These results collectively demonstrate that acute application of -15° HDT caused a reduction in CSF flow variables (systolic peak flow and peak-to-peak pulse amplitude) which, when coupled with a decrease in average cerebral arterial flow, systolic peak flow, and peak-to-peak pulse amplitude, is consistent with a decrease in cardiac-related pulsatile CSF flow. These results suggest that decreases in cerebral arterial inflow were the principal drivers of decreases in CSF pulsatile flow. Background Microgravity has been associated with multiple maladaptive physiological processes, including alterations to cerebrospinal fluid (CSF) flow and pressures in microgravity. CSF production and its flow characteristics serve as a key component of the physiological homeostasis, immunological protection, and metabolic maintenance of the central nervous system. Magnetic resonance imaging (MRI) studies in astronauts before and after spaceflight missions have shown perturbations to the CSF spaces, predominantly after long-duration missions, including narrowing of the central sulcus, upward shift of the brain, and narrowing of the CSF spaces at the vertex [1]. predominantly after long-duration missions, including narrowing of the central sulcus, upward shift of the brain, and narrowing of the CSF spaces at the vertex [1]. A spaceflight-induced condition thought to be related to alterations in CSF flow dynamics is Spaceflight Associated Neuro-ocular Syndrome (SANS), a spectrum of poorly understood neuro-ophthalmological findings documented in a subset of astronauts returning from the International Space Station. Clinical findings of SANS include unilateral and bilateral optic disc edema, globe flattening, choroidal and retinal folds, hyperopic refractive error shifts, and choroidal and nerve fiber layer thickening [2]. Of seven United Page 2/16 States astronauts evaluated for SANS after long-duration spaceflight of greater than six months, six developed decreased near field vision with nerve fiber layer thickening; five exhibited optic disc edema, globe flattening, and choroidal folds; and three had nerve fiber layer thickening [2, 3]. Four individuals who underwent postflight lumbar puncture for measurement of CSF pressure exhibited an elevated average opening pressure of 28 cm H2O up to 12 days after reentry [2, 3]. In addition, postflight surveys of 300 astronauts suggest that inflight loss of both near and distant visual acuity is a significant issue that increases in prevalence with duration of flight, with 29% of short duration and 60% of long duration mission participants reporting some degree of loss in visual acuity [2, 4]. Alterations in CSF dynamics secondary to loss of hydrostatic pressure gradients in microgravity are thought to play a role in SANS. Such alterations may include an overall mild elevation in intracranial pressure (ICP) and/or a loss of normal CSF pressure changes due to postural changes on Earth; however, the physiology of the interaction between cerebral blood flow and CSF in microgravity is not well understood. Background Although not conclusively known, the pathology observed in astronauts may suggest transiently increased ICP; according to the “pulse absorber” or “cerebral Windkessel” theory, this should correspond to an increase in CSF flow pulsation [5, 6]. Thus, both cerebral venous and arterial flow, as the principal contributors to changes in CSF flow related to this increased ICP, are physiologically plausible factors affecting CSF flow dynamics, as demonstrated in studies of young adults and elderly subjects by PCMR [7]. Cerebral arterial and jugular venous flow pulsations correlate significantly with CSF flow pulsations, with cerebral arterial flow having a predominantly positive contribution to CSF flow pulsations, opposite to the contribution of jugular venous flow [8]. Thus, a decrease in arterial cerebrovascular inflow would be associated with a decrease in cardiac-related pulsatile CSF flow. It is also notable that work by Roberts et al. suggests that the upward shift of the brain in microgravity with tissue crowding at the vertex may compress adjacent venous structures, causing obstruction of CSF and venous outflow, thereby potentially resulting in an elevation of ICP [1]. Prior studies using ground-based simulated microgravity have demonstrated changes in cerebral arterial and jugular flow dynamics, however changes in CSF pulsations would benefit from improved understanding. The study of cerebral arterial, venous, and CSF flow dynamics as they relate to microgravity would be valuable in their own right as well as being useful for helping to validate whole- body models that predict SANS pathophysiology and seek to evaluate the effectiveness of countermeasures [4, 9, 10]. Head-down tilt (HDT) has been used to simulate microgravity in ground-based studies that seek to quantify changes in arterial and venous flow dynamics [11]. However, prior studies have yielded conflicting results about arterial blood flow dynamics and have not comprehensively studied CSF flow dynamics, which is an important underlying aspect of the “fluids shift” hypothesis for SANS [4, 12, 13]. In the present study, we hypothesized that microgravity conditions simulated by acute HDT would result in increased CSF pulsatile flow. Our study was designed to analyze the effect of HDT simulated microgravity on cerebrovascular flow dynamics by measuring flow through major arteries, veins, and CSF Page 3/16 spaces using phase contrast magnetic resonance (PCMR) before and after acute application of 15° HDT in healthy volunteers. spaces using phase contrast magnetic resonance (PCMR) before and after acute application of 15° HDT in healthy volunteers. Physiological Measurements: Heart rate was monitored throughout the exam using a peripheral pulse unit applied to the first digit of the volunteer’s left hand. Blood pressure readings were taken prior to the exam, in the supine position, and in the HDT position after completion of the MRI scan. Blood pressure readings were taken with a MR-safe digital automated sphygmomanometer applied to the upper arm while the volunteer was supine. Study Design: Fifteen healthy subjects participated in this NASA and Emory University IRB-approved study protocol. This study adhered to the tenets of the Declaration of Helsinki. Criteria for inclusion in the study included age ≥18 years, no contraindications to non-contrast MRI, no self-reported cardiovascular or neurological disease, and no current pregnancy. Written informed consent was obtained for all volunteers. All data was anonymized prior to analysis. Physiological Measurements: In-Vivo MRI Measurements: All volunteers were scanned on a 3 T MRI scanner (Prisma Siemens Medical Systems, Malvern, PA) using a 20-channel head coil. Two-dimensional cine phase-contrast MRI (PCMR) images were acquired in the transverse orientation at the mid-C2 vertebral level using a previously published protocol [14]. Images were acquired with through-plane velocity encoding in the supine position and again after a 30-minute acclimation period at 15° HDT (Fig 1). Forty images were reconstructed for each cardiac cycle using retrospective peripheral pulse unit (PPU) gating. Imaging variables included a field-of-view (FOV) = 175 x 175 mm, in-plane resolution = 1.1 x 1.1 mm, slice thickness = 5.0 mm, repetition time (TR) = 11 ms, echo time (TE) = 7 ms, flip angle = 30°, and two segments. Two scans were acquired at the mid-C2 location, one with a velocity encoding (VENC) value of 80 cm/s for blood flow and one with a VENC value of 10 cm/s for CSF flow. The 15° HDT was achieved using a custom-made wedge (Fig 1). This scan protocol was performed twice for each volunteer, first while supine, and then under HDT following a 30-minute acclimatization period under HDT. To quantify CSF, venous, and arterial flow, regions of interest (ROI) were outlined within the subarachnoid space, the bilateral internal jugular veins, and the bilateral internal carotid and vertebral arteries, respectively (Fig 2). ROIs were drawn manually to approximate the contours of each vessel using FLOW software suite (LUMC, Lieden, The Netherlands) [15, 16]. Each ROI for each of the forty images per scan was visually inspected to ensure quality of the contours. The automatic segmentation feature was applied at each time-step to correctly segment the vessel wall. All ROIs were visually inspected to assure that phase aliasing was not present, and if found, phase unwrapping was applied. Page 4/16 Page 4/16 Page 4/16 Since pulsatile CSF flow was measured at the mid-C2 vertebral level, we assumed that measured pulsatile flow should sum to zero over the cardiac cycle due to the oscillatory nature of CSF flow dynamics with approximately zero net flow present in the spinal subarachnoid space over acute time scales [17, 18]. In practice, this was not observed, due to the presence of eddy currents and background phase offsets in the PCMR scan [19]. Statistical Analysis: SAS version 9.4 and Microsoft Excel were used to conduct all data analyses. Normality for all study variables was assessed with a Shapiro-Wilk test. All outcome measures apart from venous cross- sectional area passed the Shapiro-Wilk test and were therefore analyzed through parametric methods, namely by paired two-tailed t-tests (supine vs. HDT). The comparisons in these statistical tests were conducted on each individual in a paired manner, comparing before and after HDT. The statistical significance of the difference in venous cross-sectional area due to HDT was accordingly analyzed through the non-parametric Wilcoxon signed-rank test. Differences were considered significant at p-value < 0.05. In-Vivo MRI Measurements: Therefore, we specified a subject-specific flow rate offset to force the net pulsatile CSF flow to be zero over each cardiac cycle as has been applied in previous studies [20-23]. The average required offset magnitude was 0.22 ± 0.34 mL/s. Similar to previous publications, we computed CSF flow volume as half of the integral of the absolute value of pulsatile CSF flow rate over the cardiac cycle. Numerical integration was performed using the trapezoid rule (Fig 3) [24]. Instantaneous flow rates (mL/min) at 40 time points throughout the cardiac cycle were determined in the veins, arteries, and CSF space using the ROI area and corresponding average velocity measurements as determined by the FLOW program (Figs 3-4). Our primary outcome measures were: average flow rate over the cardiac cycle, systolic peak flow rate, peak-to-peak flow pulse amplitude (PtPPA), flow volume per cardiac cycle, and cross-sectional area (CSA) for arteries, veins, and the spinal subarachnoid space (Fig 3). These specific variables were chosen to allow direct comparison with prior HDT studies (average systolic peak flow, flow volume/cycle, CSA) as well as due to their prior use in describing changes in intracranial CSF pulsations (PtPPA) [6, 12, 13, 25]. These outcome measures were determined from a combination of ROIs on the PCMR cine magnitude images (providing CSA) as well as measured velocity through that area. Results Fifteen healthy subjects (7 males, 8 females; mean age ± SD: 29.5 ± 12.1 yrs.) participated in the study. Vital signs revealed no significant difference between heart rates or systolic and diastolic blood pressure before and after HDT (Table 1). Page 5/16 Page 5/16 Venous blood flow variables showed an increase in the internal jugular vein cross-sectional area from baseline to HDT (Table 2, Figure 5). In terms of arterial blood flow variables, changes in average blood flow rate, systolic peak flow rate, and PtPPAart all reached statistical significance (Table 2). Finally, CSF flow variables showed a statistically significant decrease in systolic peak flow rate and PtPPAcsf. (Table 2, Figure 6, Figure 7). Discussion Our hypothesis was that microgravity, as simulated through acute application of 15° HDT, would result in an increase in cardiac-related pulsatile CSF flow. Our results are inconsistent with this hypothesis, instead showing that HDT was significantly associated with a decrease in pulsatile CSF flow at the mid-C2 vertebral level as manifested by reduced systolic peak flow rate and PtPPAcsf. In addition, HDT was Page 6/16 Page 6/16 significantly associated with decreased cerebral arterial average flow rate, systolic peak flow rate, and PtPPAart. Finally, a significant increase in jugular venous cross-sectional area was also observed. Prior studies have examined cerebral flow dynamics under HDT. Marshall-Goebel et al., using 9 healthy male volunteers, found a decrease in both arterial and venous flow rate variables as well as an increase in venous cross-sectional area (CSA) from baseline to HDT (62 mm2 to 97 mm2), suggestive of increased venous pressure [13]. Ishida et al., using 15 healthy volunteers, found increases in venous CSA (36 mm2 to 54 mm2), decreases in arterial inflow, increases in venous outflow, and no significant changes in CSF flow volume/cycle or systolic velocity [12]. These observed changes in venous CSA under ground-based HDT are consistent with ultrasound studies performed during spaceflight that demonstrate comparable increases in venous CSA in subjects exposed to microgravity, which lends validity to the results of these HDT protocols [26, 27]. These prior studies collectively corroborate our findings of increased venous CSA and decreased arterial inflow. Finally, we observe a decrease in pulsatile CSF flow not examined in these prior HDT studies. The methods described in this work may be useful to understand CSF flow pulsatility increases that may be present after prolonged strict HDT. Our findings of increased jugular venous cross-sectional area suggest increased cerebral venous pressure. Venous pressure has been shown by Holmlund et al. to be predictive of changes in ICP with increases in venous CSA correlating with increases in ICP [28]. These findings are, however, tempered by one study that showed that measured ICP and central venous pressure both decreased during acute (<1 minute) episodes of zero G in parabolic flight, which differs from our findings of increased central venous pressure during HDT [29]. This difference may be due to the duration (<1 minute vs. longer duration HDT) or the mode (parabolic flight vs HDT) of gravitational change. Discussion Apart from the pathophysiological alterations suggested by the measurements in our study, these data are useful inputs for models seeking to simulate volume and pressure alterations in the head and eye in microgravitational settings. Prior studies have relied on blood and aqueous humor dynamics as inputs to models, and we anticipate that these results will be similarly useful for models seeking to replicate the pathophysiology of SANS [9]. The limitations of our study included the fact that our recorded venous outflow did not account for the entirety of arterial inflow, indicating missing venous collateral changes in flow. Jugular venous flow, as opposed to venous flow through other vessels such as the vertebral venous system, increases from an erect to supine position [32]. As a result, changes in body position from upright to supine to HDT could have effects on collateral venous flow that went unmeasured in our study. In addition, observed changes of flow in the jugular veins from supine to HDT in our study could plausibly be a result of the increased jugular recruitment seen in changes in body position from upright to supine [32]. We focused on the jugular veins in our study since they were the only veins that could be consistently identified in the images, but not being able to account for changes in collateral venous flow and their significance is a limitation. A primary limitation of our study was the use of HDT as a proxy for microgravity. Although HDT has been utilized in many previous studies to simulate microgravity, it cannot reproduce other spaceflight-related nongravitational factors such as alterations to fluid and electrolyte balance, cardiovascular and pulmonary function, and metabolism, and therefore the generalizability of any findings to spaceflight has these inherent limitations [11, 33, 34]. An additional limitation includes the duration of the study. As previously mentioned, ocular symptoms in astronauts increase in prevalence with duration of spaceflight, and a 30-minute HDT analog captures only acute changes. Moreover, an ideal angle for HDT has not been established, with a range of angles from 6° to 15° being used. The 15° angle used in our study may have been too steep and we cannot ensure that our CSF findings would have been replicated with a lower degree of HDT. Discussion To help interpret our CSF results, it is useful to recall several aspects of CSF physiology under normal gravity conditions, specifically the coupling of CSF and cerebral blood flow dynamics. CSF is produced in the choroid plexus of the brain’s ventricles and slowly circulates into the spinal and cranial subarachnoid spaces [30]. It is then absorbed into the venous system via arachnoid granulations, primarily near the dural venous sinus [30]. An increase in venous pressure can alter the pressure gradient across the arachnoid granulations, leading to reduced CSF absorption and an increase in total intracranial CSF volume [4]. However, a decrease in CSF absorption is not expected under acute HDT since any rise in venous pressure is likely counterbalanced by a similar rise in ICP. Our study did not directly measure change in intracranial CSF volume or compliance under acute HDT. Thus, we are not able to conclude how the observed decrease in CSF flow amplitude related to change, if any, in intracranial compliance. As noted above, CSF flow from the brain has significant contribution from cardiac related pulsation, with cerebral arterial flow pulsations having a positive contribution to CSF flow pulsations [8]. SANS may involve perturbations to CSF flow associated with mild increases in ICP given the pathological findings of optic disc edema, globe flattening, and decreased visual acuity that are also present in Earth-bound conditions of CSF imbalance such as idiopathic intracranial hypertension [2, 31]. CSF dynamics can be Page 7/16 Page 7/16 altered by changes in production, flow, or reabsorption. However, the former and latter occur slowly, and thus are unlikely to be relevant in the setting of an acute HDT study. altered by changes in production, flow, or reabsorption. However, the former and latter occur slowly, and thus are unlikely to be relevant in the setting of an acute HDT study. Collectively, these results suggest that changes in cerebral arterial flow are a plausible cause for the observed decrease in CSF pulsatile flow. Because the elevated ICP expected in HDT would be expected to increase CSF flow pulsatility, we further suggest that the observed decrease in arterial pulsatility was a dominant factor leading to the findings. Since we did not measure intracranial compliance, it is not possible to determine the relative importance of intracranial compliance change with respect to CSF flow pulsatility under acute HDT . Discussion A study using a 15° HDT protocol to assess venous jugular blood flow before, during, and after spaceflight on International Space Station crew members demonstrated that venous CSA increased from sitting to supine to HDT with a similar magnitude of change between those positions preflight and postflight, although HDT in both of those settings overestimated increases in venous CSA found in-flight Page 8/16 [35]. However, our venous and arterial changes from baseline to 15° HDT are corroborated by prior studies at these lower degrees of HDT, which lends support to the validity of our CSF findings at 15° [12, 13, 25]. [35]. However, our venous and arterial changes from baseline to 15° HDT are corroborated by prior studies at these lower degrees of HDT, which lends support to the validity of our CSF findings at 15° [12, 13, 25]. Our scan protocol did not specify a defined stabilization period prior to the initial supine MRI scan. However, each volunteer was positioned in the supine position for at least 20 minutes before the scan used for evaluating flow. An additional limitation was that, due to time constraints, we did not repeat the scan protocol afterwards again in the supine position to assess return to baseline. We also were not able to monitor CO2 during our scan protocol and so cannot correlate our findings with any such changes in a patient’s breathing while undergoing these positional changes. Finally, the radiofrequency coil required that the head be at a 0° orientation above the neck. Excessive neck flexion has been associated with increased ICP and therefore could be a potential confounder of our findings [36]. In terms of limitations in statistical analysis, we chose not to apply a Bonferonni correction for multiple comparisons (due to the multiple variables in our study) so as to not make a type II error more likely. However, a lack of correction for multiple comparisons does increase the likelihood of a type I error. As a result, chance associations may exist, and our positive findings should be replicated. A final limitation includes the lack of well-defined minimal clinically important differences (MCIDs) in our flow variables with respect to SANS. Future studies should compare arterial, venous, and CSF flow variables in astronauts pre- and post-flight, particularly in those experiencing SANS symptoms in order to generate MCIDs. Discussion Non-invasive measurements of these flow variables in astronauts during spaceflight would also be ideal if permitted by technology. In addition, comparisons of HDT variables with imaging from astronauts would help validate HDT as a tool for further investigation of physiological changes to the head in microgravity, which would be critical to justify future studies examining specific populations of interest such as women, minorities, various age groups, or even children to create more individualized risk profiles and predictive models. In conclusion, we have demonstrated that acute application of 15° HDT to simulate microgravity was associated with alterations in intracranial blood flow and spinal CSF flow dynamics, specifically a reduction in CSF pulsatile flow variables. When combined with our observed decreases in cerebral arterial flow variables as well as a significant increase in jugular venous cross-sectional area, these findings collectively suggest that decreases in cerebral arterial flow were the principal drivers of our observed decreases in CSF pulsatile flow. Declarations Acknowledgements The datasets used and analyzed during this study are available from the corresponding author on reasonable request. The datasets used and analyzed during this study are available from the corresponding author on reasonable request. Funding This work was funded by Georgia CTSA Grants TL1TR002382 and UL1TR002378, NASA grant NNX16AT06G, and the Georgia Research Alliance (CRE). This work was funded by Georgia CTSA Grants TL1TR002382 and UL1TR002378, NASA grant NNX16AT06G, and the Georgia Research Alliance (CRE). Author Contributions All authors reviewed the final manuscript. AZ collected and analyzed data and wrote the manuscript. SC contributed to data collection, figure 1, and background research and citations. BAM contributed to data analysis methods, background research, citations, and manuscript writing. JO and CRE formulated, designed, and implemented the overall project under which this specific research was conducted along with contributing to background research, citations, and manuscript writing. Competing interests BAM is an employee of Alcyone Therapeutics and has served as a consultant to Roche, Genentech, Voyager Therapeutics, Praxis Medicines, InviCRO, Biogen, Voyager Therapeutics, Behavior Imaging, Medtrad Biosystems, Neurosyntek, and Cerebral Therapeutics. The authors otherwise declare that they have no competing interests. Availability of data and materials Availability of data and materials Acknowledgements The authors would like to thank our volunteers and MRI support staff for their contributions to this study. The authors would like to thank our volunteers and MRI support staff for their contributions to this study. Page 9/16 Ethics approval and consent to participate This study was approved by both the NASA and Emory University IRBs. 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Lawley, J.S., et al., Effect of gravity and microgravity on intracranial pressure. J Physiol, 2017. 595(6): p. 2115-2127. 30. Bothwell, S.W., D. Janigro, and A. Patabendige, Cerebrospinal fluid dynamics and intracranial pressure elevation in neurological diseases. Fluids Barriers CNS, 2019. 16(1): p. 9. 31. Hu, R., et al., Cerebrospinal Fluid Pressure Reduction Results in Dynamic Changes in Optic Nerve Angle on Magnetic Resonance Imaging. J Neuroophthalmol, 2019. 39(1): p. 35-40. 32. Valdueza, J.M., et al., Postural dependency of the cerebral venous outflow. Lancet, 2000. 355(9199): p. 200-1. 33. Watenpaugh, D.E., Analogs of microgravity: head-down tilt and water immersion. J Appl Physiol (1985), 2016. 120(8): p. 904-14. 34. Hargens, A.R. and L. Vico, Long-duration bed rest as an analog to microgravity. J Appl Physiol (1985), 2016. 120(8): p. 891-903. 35. Marshall-Goebel, K., et al., Assessment of Jugular Venous Blood Flow Stasis and Thrombosis During Spaceflight. JAMA Netw Open, 2019. 2(11): p. e1915011. Page 12/16 Page 12/16 36. Williams, A. and S.M. Coyne, Effects of neck position on intracranial pressure. Am J Crit Care, 1993. 2(1): p. 68-71. 36. Williams, A. and S.M. Coyne, Effects of neck position on intracranial pressure. Am J Crit Care, 1993. 2(1): p. 68-71. Figures Figure 1 Volunteer under -15° head-down tilt in the MR scanner. Note that the head (not pictured) was positioned within a radiofrequency coil that required it to be positioned at 0° orientation above the neck. Figure 1 Volunteer under -15° head-down tilt in the MR scanner. Note that the head (not pictured) was positioned within a radiofrequency coil that required it to be positioned at 0° orientation above the neck. Volunteer under -15° head-down tilt in the MR scanner. Note that the head (not pictured) was positioned within a radiofrequency coil that required it to be positioned at 0° orientation above the neck. Page 13/16 Page 13/16 Figure 2 Figure 2 Example of CSF (image 1), arterial (image 2), and venous (image 3) ROIs as seen on an axial view at the mid-C2 vertebral level. Each scan is from a different volunteer. Left greyscale panels: phase contrast image showing craniocaudal velocities (greyscale). Right panels: anatomic image used to identify vessels and other structures. Image 1 structures: subarachnoid space (red). Image 2 structures: internal carotid (red and green) and vertebral arteries (white and blue). Image 3 structures: Internal jugular veins (red and green). Instantaneous flow rates (mL/min) at 40 time points throughout the cardiac cycle were determined in the veins, arteries, and CSF spaces using the ROI area and corresponding average velocity measurements as determined by the FLOW program. The software also provided stroke volumes for each vessel, defined as the net blood volume crossing the measurement plane per cardiac cycle. Figure 2 Example of CSF (image 1), arterial (image 2), and venous (image 3) ROIs as seen on an axial view at the mid-C2 vertebral level. Each scan is from a different volunteer. Left greyscale panels: phase contrast image showing craniocaudal velocities (greyscale). Right panels: anatomic image used to identify vessels and other structures. Image 1 structures: subarachnoid space (red). Image 2 structures: internal carotid (red and green) and vertebral arteries (white and blue). Image 3 structures: Internal jugular veins (red and green). Instantaneous flow rates (mL/min) at 40 time points throughout the cardiac cycle were determined in the veins, arteries, and CSF spaces using the ROI area and corresponding average velocity measurements as determined by the FLOW program. The software also provided stroke volumes for each vessel, defined as the net blood volume crossing the measurement plane per cardiac cycle. Figures Example of CSF (image 1), arterial (image 2), and venous (image 3) ROIs as seen on an axial view at the mid-C2 vertebral level. Each scan is from a different volunteer. Left greyscale panels: phase contrast image showing craniocaudal velocities (greyscale). Right panels: anatomic image used to identify vessels and other structures. Image 1 structures: subarachnoid space (red). Image 2 structures: internal carotid (red and green) and vertebral arteries (white and blue). Image 3 structures: Internal jugular veins (red and green). Instantaneous flow rates (mL/min) at 40 time points throughout the cardiac cycle were determined in the veins, arteries, and CSF spaces using the ROI area and corresponding average velocity measurements as determined by the FLOW program. The software also provided stroke volumes for each vessel, defined as the net blood volume crossing the measurement plane per cardiac cycle. Page 14/16 Page 14/16 Figure 3 Representative CSF flow rate vs. time over a cardiac cycle, showing the definitions of flow and pulsatility variables. Note that negative flow rates present during systole are in the craniocaudal direction. The pulse was monitored by a peripheral pulse unit applied to the first digit of the volunteer’s hand, and the local systolic flow peak occurs earlier at the vessels of the neck than at the first digit, explaining the location of the systolic peak in this figure. Time zero of the cardiac cycle in this figure represents the detected pulse peak at the finger. Page 15/16 p g Figure 4 Figure 4 Page 15/16 Representative arterial and venous flow rates vs. time over a cardiac cycle for a single subject for the right internal carotid artery and the right internal jugular vein. Negative values indicate flow towards the heart. The explanation for the late location of the systolic peak in this figure is the same as for Figure 3. Page 16/16
https://openalex.org/W2111851901	https://europepmc.org/articles/pmc3279502?pdf=render	English	null	Correlation Between High-sensitivity C-reactive Protein and Reactive Oxygen Metabolites During A One-year Period Among Asymptomatic Subjects	Journal of clinical medicine research	2,012	cc-by	2,333	J Clin Med Res • 2011;4(1):52-55 ress Elmer J Clin Med Res • 2011;4(1):52-55 ress Elmer Short Communication J Clin Med Res • 2011;4(1):52-55 Abstract Background: Inflammation and oxidative stress are associated with human health and the disease status. The present study aimed to investigate the longitudinal correlation between the diacron re- active oxygen metabolites (d-ROMs) level, as an oxidative stress- related marker, and high-sensitivity C-reactive protein (hsCRP), as an inflammatory marker, during a one-year period among asymp- tomatic subjects. Correlation Between High-sensitivity C-reactive Protein and Reactive Oxygen Metabolites During A One-year Period Among Asymptomatic Subjects Kazuhiko Kotania, b, Nobuyuki Taniguchia Keywords: Oxidative stress; Oxygen reactive species; Inflamma- tion; CRP Introduction In recent years, chronic inflammation, as assessed by the C-reactive protein (CRP) level, and oxidative stress, as assessed by oxidative stress-related markers, have attracted much attention as non-traditional pathological factors, since these conditions have been shown to play important roles in human health and disease [1-5]. While the measurement of high-sensitivity CRP (hsCRP) in blood is currently commonplace for evaluating inflammation, the oxidative stress-related markers that can be used to easily evaluate the oxidative stress status in the clinical settings remain under consideration [6]. Methods: The data, including anthropometric and biochemical markers, were collected at baseline and after the one-year period from 71 participants (male/female = 41/30, mean age 50 years). The correlation between the changes of the d-ROMs and hsCRP levels during the study period was examined. Results: A simple correlation analysis showed a significant and positive correlation to exist between the changes of the d-ROMs and hsCRP levels (r = 0.40, P < 0.01). This significant correlation remained independent in a multiple linear regression analysis ad- justed for confounding factors. The diacron reactive oxygen metabolites (d-ROMs) test was recently introduced, and this measurement can quantify the oxidative stress status by measuring the levels of hydro- peroxides of global organic compounds (lipids, proteins, nu- cleic acids, etc.) [7, 8]. This test is currently used as an easy clinical marker for evaluating oxidative stress. In fact, a few studies have reported a significantly positive relationship be- tween the d-ROMs and hsCRP levels in patients at high risk for coronary artery disease [9] and in general populations [10-12]. However, these studies were based on a cross-sec- tional design; therefore, prospective studies are required to confirm their relationships. The aim of the present study was thus to investigate the longitudinal correlation between the d-ROMs and hsCRP levels in an asymptomatic population. Conclusions: The present findings suggest that the relationship between the d-ROMs and hsCRP levels could be prospectively fol- lowed, and that monitoring both markers may help to better un- derstand the cooperation of inflammation and oxidative stress in association with health and disease. Further studies are necessary to clarify the biological mechanism(s) responsible for the observed relationship. Manuscript accepted for publication Noveber 11, 2011 aDepartment of Clinical Laboratory Medicine, Jichi Medical University, Shimotsuke-City, Tochigi, Japan bCorresponding author: Department of Clinical Laboratory Medicine, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke-City, Tochigi, 329-0498, Japan. Email: kazukotani@jichi.ac.jp doi:10.4021/jocmr755w Methods This one-year observational design study consisted of 71 asymptomatic subjects (male/female = 41/30, mean 50 ± 7 years) who had health check-up examinations both at base- line and after the one-year period. The inclusion criteria were non-smokers and participants not taking any medications or 52 Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org J Clin Med Res • 2011;4(1):52-55 Kotani et al Table 1. The Correlation Between the Changes of the D-ROMs and HsCRP Levels During A One-year Period D-ROMs: diacron reactive oxygen metabolites; HsCRP: high-sensitivity C-reactive protein; HDL: high- density lipoprotein. r: simple correlation test (Pearson’s test), β: multiple linear regression analysis, ad- justed for all the measured factors. The triglycerides and hsCRP values were log-transformed because of their skewed distributions. Significance level: P < 0.05. D-ROMs (dependent factor) HsCRP (dependent factor) r (P) β (P) r (P) β (P) Age, years 0.13 (0.30) 0.13 (0.31) 0.18 (0.14) 0.18 (0.15) Gender, male 0.01 (0.98) -0.01 (0.93) 0.05 (0.69) 0.03 (0.83) Body mass index, kg/m2 0.25 (0.04) 0.11 (0.42) 0.33 (0.01)* 0.24 (0.06) Mean blood pressure, mmHg 0.23 (0.06) 0.20 (0.12) 0.13 (0.29) 0.04 (0.73) Total cholesterol, mmoL/L -0.12 (0.31) -0.09 (0.47) -0.11 (0.35) -0.06 (0.67) HDL cholesterol, mmoL/L -0.25 (0.03)* -0.13 (0.33) -0.28 (0.02)* 0.14 (0.30) Triglyceride, mmoL/L 0.01 (0.96) -0.09 (0.48) 0.10 (0.43) 0.01 (0.97) Plasma glucose, mmoL/L 0.13 (0.27) 0.09 (0.43) 0.09 (0.48) 0.02 (0.89) HsCRP, mg/dL 0.40 (< 0.01)* 0.27 (0.04)* − − D-ROMs, U. Carr. − − 0.40 (< 0.01)* 0.26 (0.04)* Table 1. The Correlation Between the Changes of the D-ROMs and HsCRP Levels During A One-year Period ble 1. The Correlation Between the Changes of the D-ROMs and HsCRP Levels During A One-year i d D-ROMs: diacron reactive oxygen metabolites; HsCRP: high-sensitivity C-reactive protein; HDL: high- density lipoprotein. r: simple correlation test (Pearson’s test), β: multiple linear regression analysis, ad- justed for all the measured factors. The triglycerides and hsCRP values were log-transformed because of their skewed distributions. Significance level: *P < 0.05. supplements. The exclusion criteria were participants who had acute infectious diseases, such as a common cold, or who had a history of cardio/cerebrovascular, thyroid, colla- gen, severe kidney or liver diseases. This study was approved by the Institutional Ethics Committee, and all subjects gave their informed consent. between at baseline and after the one-year period were compared using paired t-tests. Methods Changes in the respective measurements during the one-year period were calculated by subtracting the values at baseline from the values after the one-year period. A simple correlation test (Pearson’s test) and a multiple linear regression analysis, adjusted for all the other factors, were used to observe the correlation between the changes of the d-ROMs and hsCRP levels during the one- year period. The TG and hsCRP values were log-transformed in these analyses because of their skewed distributions. Statistical significance was defined as a P-value < 0.05. At baseline and after the one-year period, we obtained data from each subject in an overnight fasted state. The body mass index (BMI) was calculated based on the weight and height measured while subjects wore light clothing without shoes. The systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured in the subject’s right arm with a mercury sphygmomanometer while the subject was the seated position, and then, the mean blood pressure (MBP) was calculated by the equation of DBP + [(SBP-DBP)/3]. The serum total cholesterol (TC), triglyceride (TG), HDL-C and fasting plasma glucose (FPG) levels were measured by standard enzymatic methods. The serum hsCRP levels were measured by an enzyme-linked immunosorbent assay (Assaypro Co. Ltd., St. Charles, MO, USA). The d-ROMs levels were measured using a kinetic spectrophotometric assay (the F.R.E.E system; Diacron Srl., Grosseto, Italy) with intra- and inter-assay coefficients of variation of 2.1% and 3.1%, respectively [7, 8]. Briefly, serum samples were mixed with a buffered solution, and a chromogenic substrate was added to the mixture. The mixture was centrifuged, and then incubated in a thermostatic block. The absorbance was recorded at 505 nm. The measurements are expressed in U. Carr., where 1 U. Carr. corresponds to 0.08 mg/dL H2O2. Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org References The present study found that there was an independent, sig- nificant and positive correlation between the changes of the d-ROMs and hsCRP levels during a one-year period among asymptomatic subjects. Although the cooperation of oxida- tive stress and inflammation is a recent key concept devel- oped as a result of a deeper understanding of the pathophysi- ology of health and disease [1-5], the correlation between the d-ROMs and hsCRP levels has so far been examined only in cross-sectional studies [9-12]. From the viewpoint of estab- lishing evidence, it is valuable to note that the present pro- spective study confirmed the coexistence of oxidative stress and inflammation, even though the longitudinal period of the study was relatively short. 1. Vincent HK, Taylor AG. Biomarkers and potential mechanisms of obesity-induced oxidant stress in hu- mans. Int J Obes (Lond). 2006;30(3):400-418. 2. Inoue N. Vascular C-reactive protein in the pathogenesis of coronary artery disease: role of vascular inflammation and oxidative stress. Cardiovasc Hematol Disord Drug Targets. 2006;6(4):227-231. 3. Navab M, Gharavi N, Watson AD. Inflammation and metabolic disorders. Curr Opin Clin Nutr Metab Care. 2008;11(4):459-464. 4. Cachofeiro V, Goicochea M, de Vinuesa SG, Oubina P, Lahera V, Luno J. Oxidative stress and inflammation, a link between chronic kidney disease and cardiovascular disease. Kidney Int Suppl. 2008(111):S4-9.l Although the detailed biological mechanism (s) underly- ing the present results remain unclear, there are several pos- sible explanations. Oxidative stress, which results from an imbalance in the oxidative/antioxidative system (i.e., caused by an unhealthy lifestyle), and inflammation can both induce an oxidative stress response and inflammatory molecules via cell dysfunction in systemic organs, including the adipose tissue, liver and vasculature [1, 3]. Furthermore, the path- ways form a vicious cycle of oxidative stress and inflamma- tion [13]. The oxidative stress-inflammation inter-relation- ship is even present in asymptomatic subjects, and this may partially explain the correlation between the d-ROMs and hsCRP levels. 5. Jensen GL. Inflammation: roles in aging and sarcopenia. JPEN J Parenter Enteral Nutr. 2008;32(6):656-659. 6. Stephens JW, Khanolkar MP, Bain SC. The biological relevance and measurement of plasma markers of oxida- tive stress in diabetes and cardiovascular disease. Ath- erosclerosis. 2009;202(2):321-329. 7. Iamele L, Fiocchi R, Vernocchi A. Evaluation of an auto- mated spectrophotometric assay for reactive oxygen me- tabolites in serum. Clin Chem Lab Med. 2002;40(7):673- 676. 8. Vassalle C. Results After the one-year period, the hsCRP levels showed a small but significant increase [0.026 (0.013 - 0.049) to 0.031 (0.017 - 0.068) mg/dL; P = 0.03], while the d-ROMs levels showed a non-significant and slight increase (311 ± 45 to 317 ± 47 U. Carr.; P = 0.15). The mean/median levels of the other measured factors between the values at baseline and post-one-year period were largely unchanged: with changes from 22.5 ± 3.0 to 22.6 ± 3.1 kg/m2 in the BMI (P = 0.29), 93 ± 13 to 93 ± 11 mmHg in MBP (P = 0.52), 5.32 ± 1.33 to 5.25 ± 0.91 mmol/L in TC (P = 0.51), 1.61 ± 0.41 to 1.62 ± 0.43 mmol/L in HDL-C (P = 0.42), 0.92 (0.71-1.39) to 1.02 (0.67-1.49) mmol/L in TG (P = 0.59), 5.38 ± 0.54 to 5.52 ± 0.77 mmol/L in FPG (P = 0.06). A simple correlation analysis showed that there was a significant and positive correlation between the changes of the d-ROMs and hsCRP levels (Table 1). The changes of the 2 2 The data are expressed as the means ± standard deviation (SD) or the medians plus the interquartile range. The data Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org 53 J Clin Med Res • 2011;4(1):52-55 HsCRP and D-ROMs During A One-year Period lying the observed relationship. lying the observed relationship. d-ROMs or hs-CRP levels were significantly correlated with those of the BMI (positively) or of HDL-C (inversely). A subsequent multiple linear regression analysis revealed that there remained a significant and positive correlation between the changes of the d-ROMs and hsCRP levels, independent of the changes of the other measured factors. Conflict of Interest All authors declare that they have no conflict of interest with regard to this work. 12. Sugiura T, Dohi Y, Takase H, Yamashita S, Tanaka S, Kimura G. Increased reactive oxygen metabolites is associated with cardiovascular risk factors and vascular endothelial damage in middle-aged Japanese subjects. 13. Higashi Y, Noma K, Yoshizumi M, Kihara Y. Endothe- lial function and oxidative stress in cardiovascular dis- eases. Circ J. 2009;73(3):411-418. Vasc Health Risk Manag. 2011;7:475-482. Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org Vasc Health Risk Manag. 2011;7:475-482. 13. Higashi Y, Noma K, Yoshizumi M, Kihara Y. Endothe- lial function and oxidative stress in cardiovascular dis- eases. Circ J. 2009;73(3):411-418. References An easy and reliable automated method to estimate oxidative stress in the clinical setting. Methods Mol Biol. 2008;477:31-39. There were some limitations to the present study. The observational period was relatively short, while this can have merit because it can reduce the impact from the fluctuation of various factors influencing inflammation and oxidative stress (e.g., weight cycling). The sample size was small, and the study population was basically restricted to the non-se- vere diseased participants. Future studies with a long-term prospective design and with larger and more varied popula- tions are needed.i 9. Kamezaki F, Yamashita K, Kubara T, Suzuki Y, Tanaka S, Rkouzuma R, Okazaki M, et al. Derivatives of reactive oxygen metabolites correlates with high-sensitivity C- reactive protein. J Atheroscler Thromb. 2008;15(4):206- 212. 10. Sakane N, Fujiwara S, Sano Y, Domichi M, Tsuzaki K, Matsuoka Y, Hamada T, et al. Oxidative stress, inflam- mation, and atherosclerotic changes in retinal arteries in the Japanese population; results from the Mima study. Endocr J. 2008;55(3):485-488. In summary, the present study confirmed that there was an independent, significant and positive correlation between the changes of the d-ROMs and hsCRP levels during a one- year period among asymptomatic subjects. The relationship between the d-ROMs and hsCRP levels was prospectively followed, and monitoring both markers may help to better understand the cooperation of inflammation and oxidative stress in association with health and disease. Further studies are necessary to clarify the biological mechanism (s) under- 11. Hirose H, Kawabe H, Komiya N, Saito I. Relations be- tween serum reactive oxygen metabolites (ROMs) and various inflammatory and metabolic parameters in a Jap- anese population. J Atheroscler Thromb. 2009;16(2):77- 82. Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org 54 Kotani et al J Clin Med Res • 2011;4(1):52-55 J Clin Med Res • 2011;4(1):52-55 55 55 Articles © The authors \| Journal compilation © J Clin Med Res and Elmer Press™ \| www.jocmr.org
https://openalex.org/W4328094374	https://bcpublication.org/index.php/BM/article/download/3965/3865	English	null	The collision of theCOVID-19 and the Chinese automotive industry	BCP business & management	2,023	cc-by	2,914	The collision of theCOVID-19 and the Chinese automotive industry Zihao Zhou1,* 1ESCP Business School, Turin, Province 10134, Italy. *Corresponding author email: zihao.zhou@edu.escp.eu. Abstract. In the context of the slow or even stagnant development of China's auto industry in the epidemic environment, the specific hazards caused by the epidemic are studied. A survey on the share of auto production by region, auto production, sales trends, and offline events is conducted, and the critical situation faced by the supply chain and offline marketing model is reported. It is proposed that the supply chain should change its previous strategy to increase the resilience of the supply chain against uncertain external factors; that it should shift the focus of the supply chain to the area of core components and enhance the production level of the relevant segments; that it should propose a more automated and modern production process; and that it should transform its primary marketing model from offline to online apps and live promotions. In the face of the impact of the COVID-19 on the market, the automotive industry must establish a more resilient supply chain model and a new sales model to cope with this massive market shift. Key words: supply chain, online sales, live streaming, automotive industry, auto components. BCP Business & Management Volume 38 (2023) BCP Business & Management Volume 38 (2023) EMFRM 2022 1. Introduction The surprise attack of the new crown pneumonia in 2020 nearly paralyzed the interlocking automotive industry chain. Today, domestic and international car companies have suffered varying degrees of blow and loss due to the epidemic's impact on the supply chain and other aspects. The automotive supply chain is facing a major test as the "just-in-time, best-in-stock" parts companies have been dealt a blow. The supply chain of the automotive industry is facing a significant transformation at this time of the epidemic and needs to abandon its previous model and focus in favor of a robust, resilient, and risk-resistant strategic solution; the primary marketing model and the channel through which customers receive information also needs to shift from offline to online. China's automotive industry is vast; for example, in 2021, the total value of the automotive industry in Shanghai will be RMB 758.6 billion, accounting for 17.5% of Shanghai's GDP. The situation of the automotive industry is therefore closely related to the national output. Furthermore, with Tesla's Shanghai factory producing 470,000 units per year in 2021, almost half of its global production, the ups and downs of the auto industry's output have a profound impact on the economic situation in China and the world. 2.1 Supply Chain There are three parts of the automotive supply chain to focus on, the demand side, the supply side, and the logistics side. The disruption of the supply chain due to the epidemic and the blockage of international logistics corridors has not only reduced the supply capacity of cars but also led to a sharp drop in consumption capacity and a further reduction in demand for cars in the areas affected by the epidemic. While the intelligent electric era of the automobile has added many new applications of hardware and software, components are essential to the production of vehicles. It is estimated that a typical conventional car consists of approximately 20,000 parts, and high-end cars can have upwards of 30,000 individual parts. Electric vehicle powertrains are changing, and while the mechanical structure is more simplified than fuel power, systems such as intelligent driving, thoughtful networking, and triboelectric control are also being added, increasing the use of many software and 1772 BCP Business & Management Volume 38 (2023) EMFRM 2022 hardware. Furthermore, due to the precision of the car's design, the absence of any one part can lead to the removal of the entire vehicle from the production line. hardware. Furthermore, due to the precision of the car's design, the absence of any one part can lead to the removal of the entire vehicle from the production line. The epidemic has led to a shortage in the supply of vehicles and their components, which could lead to disruptions in the automotive supply chain in some parts of the world. The supply of automotive parts and production of vehicles has been drastically reduced, and some supplies have even been disrupted due to factors such as the control of road transport in several prominent automotive production areas and the reduced speed of import and export customs clearance, and there may be a risk of disruption in the supply chain of some vehicles around the world. The renowned auto parts group Bosch Group of Germany has already warned of global supply chain disruptions, and Hyundai Motor Group has announced a complete shutdown of its Korean plant due to disruptions in the supply of parts in China[1]. 2.1 Supply Chain pp y p Since March this year, domestic auto production and consumption have been affected simultaneously due to the epidemic, while tightening epidemic controls and poor logistics around the country has become another critical issue limiting production in the auto chain [2].The main cities on the production side, Jilin and Shanghai, were affected by the epidemic. 2021 saw Shanghai and Jilin rank second and third in vehicle production, with a combined output of 2.25 million units, accounting for 21% of national production. Moreover, Tesla's Shanghai factory will produce 470,000 units per year in 2021, accounting for almost half of its global production [3].Shanghai and the surrounding area are home to not only well-known Chinese and foreign vehicle manufacturers but also numerous component companies. Core component companies are distributed accordingly in their headquarters locations, so core component companies in Shanghai and other areas have a radiating impact on the industry due to production cuts and shutdowns due to the epidemic. From the beginning to the end of March 22, FAW Group, BMW, SAIC system factories, and Tesla in the whole vehicle have been shutting down production[4]; among the component companies, wiring harness manufacturers such as Ampofo, located in Shanghai, have announced the suspension of production. At the same time, the suspension of production and logistics disruptions in the Yangtze River Delta region also affected the production of cars nationwide and even the supply overseas[5] On May 6, the China Association of Automobile Manufacturers (CAAM) projected, based on weekly data reported by key enterprises, that in April 2022, the auto industry sales (wholesale sales) were expected to complete 1,171,000 units, down 47.6% sequentially and 48.1% year-on-year; affected by the Shanghai epidemic, SAIC's April production and sales both ended with a significant dip. Among them, SAIC's vehicle production in April was 158,000 units, down 62% year-on-year; vehicle sales were 167,000 units, down 60.3% year-on-year. BYD delivered 106,042 units in April, up 313% year-on-year and 2% sequentially, a rare feat in a headwind, mainly due to BYD's adoption of in-house production of core components and a high level of vertical integration in the supply chain. The impact of unexpected events of different scales and frequencies on the automotive supply chain cannot be precisely predicted, let alone avoided. However, brands can still adjust their product mix to reduce losses. 2.2 Sales obstacles In addition, the epidemic has made sales in the automotive and automotive aftermarket more difficult. A large number of car dealers have suspended their operations, and activities such as car shows and promotions have been forced to be postponed. For the passenger car market, consumers will be cautious in choosing public travel modes in the future; several potential consumers of private cars, especially in the central and western regions where household car ownership is relatively low, will see an accelerated short-term release of demand for private cars. At the same time, the low willingness of the population to travel during and for a more extended period after the epidemic will also reduce the demand and supply of net cars and will affect the sales of new energy vehicles mainly to rental and net car customers. The automotive aftermarket, especially auto finance and auto leasing, will also experience varying degrees of decline due to reduced auto sales and lower travel intentions. Passenger car market retail sales continued to remain low, which was affected by factors such as the epidemic. 2022 April dealership turnover in many areas was affected; there are some brands, about 20% of the dealership shop customer flow disruption of the loss of large, coupled with the loss of some brand models out of supply caused a decline of more than 95%, so April passenger car retail losses[7].This year, the rhythm of the brands faced an overall adjustment, which was not conducive to the effect of local auto shows, causing certain losses to the promotion link in dealer marketing communications, and the sales environment of conventional fuel vehicles was hampered; while new energy vehicles still relied on streaming media to maintain customer enthusiasm, which was beneficial to the sales of new energy vehicles [8]. In the context of the current epidemic situation, it is increasingly important to get one's brand into consumers' initial opt-in lists through targeted online marketing activities. This is because even if the epidemic subsides, it is likely that the previous standard decision-making process will change: consumers may no longer be inclined to visit multiple brand shops to learn about models actively but will instead turn to online channels. In fact, according to our research, online channels have become the main channel for consumers to actively access information, with non-vertical online channels growing the fastest. 2.1 Supply Chain The automotive industry needs to move away from its traditional supply chain strategy of cost and efficiency optimization and instead focus on increasing supply chain resilience as a core objective. During the past two years of recurring outbreaks in other parts of the world, some foreign companies have taken the lead in transforming their supply chains to make them more resilient and sustainable. In the short term, contingency policies can be used to minimize losses. In the medium term, planning can be undertaken to prevent possible hardship, and in the long term, a new supply chain strategy system can be developed[6]. In addition, companies should actively engage in the upstream core components industry. In order to build a highly resilient supply chain, automotive enterprises can consider changing their original outsourcing strategy and more actively lay out the upstream core components to enhance the level of relevant link capabilities and improve the industry chain's voice. At the same time, more dynamic management of procurement and intervention strategies for each category can be developed. In the case of automotive semiconductors, for example, car companies have long followed the traditional value chain model, which has become dysfunctional in the context of the "lack of core." Different 1773 BCP Business & Management Volume 38 (2023) EMFRM 2022 automotive companies should explore the possibility of participating in the semiconductor value chain, taking into account their business realities, and be more proactive in meeting the challenge of "securing" the supply of automotive semiconductors. automotive companies should explore the possibility of participating in the semiconductor value chain, taking into account their business realities, and be more proactive in meeting the challenge of "securing" the supply of automotive semiconductors. o necessary to create more modern and automated production processes. It is also necessary to create more modern and automated production proces 1. Improve product design to create products that are "easier to manufacture and more suitable for automated manufacturing." 1. Improve product design to create products that are "easier to manufacture and more suitable for automated manufacturing." 2. opening up a digital thread linking multiple systems to enable multi-dimensional information binding and traceability throughout the production process. 2. opening up a digital thread linking multiple systems to enable multi-dimensional information binding and traceability throughout the production process. 3. 2.1 Supply Chain deploying a process production and logistics automation system that can switch quickly and adaptively and realize safe real-time interconnection and one-key production change between the equipment and between equipment and information systems to realize the uncrewed operation of the whole process. 2.2 Sales obstacles As such, automakers should develop a comprehensive online touchpoint strategy to optimize the effective reach of information to end customers significantly. In addition, given the epidemic's impact on the overall product launch cycle (from marketing to production), automakers need to explore more flexible and agile launch methods and consider forms such as live online launch events in addition to traditional auto shows. For example, a Shanghai-based car company completed the launch of its high-end intelligent electric car in the form of an online launch event during the epidemic closure. As the online launch did not have the limitations of an offline venue, the car company effectively communicated its product positioning with a focus on handling through a pre-recorded racing video[9]. Of course, even with an online launch, car companies still need to consider issues such as venue, on-site staff, and vehicle scheduling, the difficulty of which should not be underestimated in a fully controlled situation; however, preparing multiple sets of model launches may be a move that car companies should consider for some time to come. 1774 BCP Business & Management Volume 38 (2023) EMFRM 2022 Sales and service end: Given the unique product attributes of cars, test drives and other links are indispensable. Although car marketing and new car delivery cannot be 100% contactless, the multichannel model is still an effective means of dealing with the epidemic[10]. On the one hand, through channels such as live streaming and expanding the functionality of the official app, car companies were able to maintain uninterrupted sales and service during the epidemic. For example, an up-and-coming car brand ensured that the new car experience and communication campaign ran properly through the UGC content in its highly engaged official APP. On the other hand, by the multichannel and especially the direct connection model, some car companies were able to grasp and interact with their customer touch-points directly. At the same time, long-term brand loyalty was created through snowy customer care during the epidemic. For example, during the epidemic, the Shanghai-based sales and service team of a new car manufacturer maintained close communication with customers located in Shanghai and dropped off supplies for those in need of help, significantly enhancing customer satisfaction, public support for the brand and brand loyalty of a group of customers. 3. Conclusion In response to the impact of the pneumonia epidemic on the automotive industry, it is necessary for automotive and its components enterprises to build and improve the new retail model of automobiles and enhance the stability of their production and sales models. The epidemic has exposed the shortcomings of the physical auto distribution system. Automotive enterprises should actively participate in the new retail model of auto e-commerce platforms and integrated e-commerce platforms that integrate technologies such as big data, virtual reality, and artificial intelligence to improve the existing distribution system of physical 4S shops. Domestic automotive and parts enterprises should always maintain a sense of crisis, continuously improve the competitiveness of their own supply chains, enhance their stickiness to the upstream and downstream of the industry chain and reduce the risk of being replaced by automotive manufacturing and parts suppliers in Southeast Asia, Mexico and other regions through initiatives such as strengthening R&D cooperation with upstream and downstream enterprises, sharing demand forecast information and cooperative inventory management or seller-managed inventory. The global automotive industry chain may face major layout adjustments in the coming period, and the auto sales model may also be reshaped at an accelerated pace. The proportion and position of China's auto industry in the global auto manufacturing supply chain may be affected to a certain extent, but this is also a new opportunity for China's auto industry to transform and upgrade and enhance its competitiveness. Every major crisis in history has often been accompanied by accelerated upgrading and management innovation; the best companies will eventually emerge, and the industrial structure of China's automotive industry will be further adjusted and optimized. [8] Fudan Research Institute: "Every major crisis in history has been accompanied by accelerated industrial upgrading and management model innovation."(2022) [9] Guan Mingyu,McKinsey & Company, "Overcoming the challenges together: China's auto industry under the "big test" of the COVID-19," (2022) [10] Ipsos, “Impact of Coronavirus to new car purchase in China,2020 [10] Ipsos, “Impact of Coronavirus to new car purchase in China,2020 BCP Business & Management EMFRM 2022 Volume 38 (2023) [1] IHS Markit,2022 [1] IHS Markit,2022 [2] Russell Flannery, ‘Gigantic’ Impact From China Lockdowns To Hit Auto Industry Globally: Automobility’s Bill Russo,2022, [2] Russell Flannery, ‘Gigantic’ Impact From China Lockdowns To Hit Auto Industry Globally: Automobility’s Bill Russo,2022, [3] Fudan Research Institute: "Every major crisis in history has been accompanied by accelerated industrial upgrading and management model innovation."(2022) [3] Fudan Research Institute: "Every major crisis in history has been accompanied by accelerated industrial upgrading and management model innovation."(2022) [4] Simon Leplâtre, “China's automotive sector crumbling under the weight of the zero-Covid policy”,202 [6] Peng he,This industry was crippled by the coronavirus - here's how it's fighting back,2020 [7] Mengze Gong, “Investigation of the current situation of the automotive industry chain under the epidemic: the production of parts according to the situation of small and medium-sized suppliers face the risk of "collapse”,2022 [7] Mengze Gong, “Investigation of the current situation of the automotive industry chain under the epidemic: the production of parts according to the situation of small and medium-sized suppliers face the risk of "collapse”,2022 1775 BCP Business & Management Volume 38 (2023) EMFRM 2022 1776
https://openalex.org/W2265035095	https://www.nature.com/articles/srep21666.pdf	English	null	NATpipe: an integrative pipeline for systematical discovery of natural antisense transcripts (NATs) and phase-distributed nat-siRNAs from de novo assembled transcriptomes	Scientific reports	2,016	cc-by	5,278	NATpipe: an integrative pipeline for systematical discovery of natural antisense transcripts (NATs) and phase-distributed nat-siRNAs from de novo assembled transcriptomes received: 08 December 2015 accepted: 28 January 2016 Published: 09 February 2016 Nat-siRNAs (small interfering RNAs originated from natural antisense transcripts) are a class of functional small RNA (sRNA) species discovered in both plants and animals. These siRNAs are highly enriched within the annealed regions of the NAT (natural antisense transcript) pairs. To date, great research efforts have been taken for systematical identification of the NATs in various organisms. However, developing a freely available and easy-to-use program for NAT prediction is strongly demanded by researchers. Here, we proposed an integrative pipeline named NATpipe for systematical discovery of NATs from de novo assembled transcriptomes. By utilizing sRNA sequencing data, the pipeline also allowed users to search for phase-distributed nat-siRNAs within the perfectly annealed regions of the NAT pairs. Additionally, more reliable nat-siRNA loci could be identified based on degradome sequencing data. A case study on the non-model plant Dendrobium officinale was performed to illustrate the utility of NATpipe. Finally, we hope that NATpipe would be a useful tool for NAT prediction, nat-siRNA discovery, and related functional studies. NATpipe is available at www.bioinfolab. cn/NATpipe/NATpipe.zip. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 08 December 2015 accepted: 28 January 2016 Published: 09 February 2016 Scientific Reports \| 6:21666 \| DOI: 10.1038/srep21666 An integrative pipeline for NAT and phased nat-siRNA discovery An integrative pipeline for NAT and phased nat-siRNA discovery In this study, by integrating several existing programs such as BLAST20, RNAplex21 and Bowtie22, we developed a pipeline named NATpipe, allowing users to do a systematical search for the NATs in the organisms without reference genomes. More importantly, following NAT predictions, the pipeline enables users to identify the phase-distributed nat-siRNA loci within the perfectly annealed regions of the NAT pairs based on sRNA and degradome sequencing data (Fig. 1). Considering the lack of the reference genomes, the criteria previously used for trans-NAT prediction11 were adopted by our pipeline. The prediction starts from BLAST search (see user man- ual in Data S1 for parameter setting), treating the de novo assembled transcripts as the input data. Based on the BLAST results, the predicted NATs will be classified as “HC” (high coverage; the complementary region is longer than 50% of either transcript of the NAT pair) or “100-nt” (the consecutive complementary region of the NAT pair should be 100 nt or longer). Then, RNAplex (see user manual in Data S1 for parameter setting) is employed to verify the annealing potential of the BLAST-predicted NAT pairs at the secondary structure level. After com- parison between the BLAST- and the RNAplex-derived results, the NATs fulfilling the two criteria are retained for further analysis: (1) the RNAplex-predicted annealed region of the NAT pair should overlap at 80% or more with the BLAST-predicted complementary region; (2) for the NAT pair predicted by RNAplex, any bubble within the annealed region should be no larger than 10% of this region. As a result, a NAT list along with the detailed information of the hybridized transcripts and their annealed regions will be available for the users. According to the RNAplex prediction, if a NAT pair has a single perfectly annealed region, it will be regarded as a cis-NAT pair candidate albeit the lack of the genomic information. g Nat-siRNAs were reported to be implicated in regulating gene expression through target cleavages or chroma- tin modifications in both animals and plants1,4,7,13–19. On the other hand, the annealed regions of the NAT pairs are the hotspots for the generation of nat-siRNAs with variable sequence length7,16,23–30. However, we recognized that it would not make any sense for the biologists just by showing them the hotspots of siRNA loci based on sRNA sequencing data. www.nature.com/scientificreports/ www.nature.com/scientificreports/ (PHO1;2) and cis-NATPHO1;2. The intriguing finding is that cis-NATPHO1;2 has an unexpected role in pro- moting the translation of its complementary partner PHO1;2, which could affect the phosphate homeostasis of rice2. Systematical study on long non-coding natural antisense transcripts (lncNATs) in Arabidopsis uncovered a total of 37,238 NAT pairs. Hundreds out of these NAT pairs exhibited light-responsive expression patterns which were potentially resulted from histone acetylation on the NAT gene loci3. In animals, NATs have been proposed to be implicated in alternative splicing of pre-mRNAs, microRNA (miRNA) binding site mimicking, chromatin remodeling, and double-stranded RNA-dependent processes such as RNA editing and RNA interference4–7.if g p p g In view of the biological significance of the NATs, several research groups have made great efforts to set fea- sible criteria for computational identification of NAT pairs from genome or transcriptome sequencing data3,8–10, and the NAT databases have been established for both animals and plants11,12. AntiHunter 2.0 is a bioinformatics tool for fast and sensitive extraction of antisense transcripts from BLAST outputs8. Unfortunately, it was designed for EST (expressed sequence tag) sequencing data, and the maximum query size was limited to 3 MB. Moreover, AntiHunter 2.0 is currently unavailable online (http://bioinfo.crs4.it/AH2.0). For the model plant Arabidopsis, both cis- and trans-NATs have been systematically predicted3,10. However, although the criteria for NAT predic- tion are provided in the previous reports, the computational programs are not available for researchers. As men- tioned above, many organisms do not possess reference genomes. Thus, developing a program for NAT prediction for the non-model organisms might be more anxious for the scientists not competent for programming. Followed by NAT identification, searching for the nat-siRNAs becomes a major task since many NATs exert biological roles through siRNA-guided target cleavages or chromatin modifications1,4,7,13–19. To our knowledge, no integrative pipeline has been available for both NAT prediction and nat-siRNA identification. Although some of the NAT databases, such as PlantNATsDB11, provide graphic view of the small RNA (sRNA) loci within the annealed regions of the NAT pairs, they do not provide users with detailed genomic arrangement of these sRNA loci and further evidences for extracting reliable nat-siRNA candidates. Brief introduction of NATs (natural antisense transcripts) and nat-siRNAs (small interfering RNAs originated from NATs) g g ) Natural antisense transcripts (NATs) are pairs of complementary transcripts encoded by the endogenous genes of plants or animals. The NATs could be protein-coding or non-protein-coding (non-coding) transcripts. Relying on the high complementarity of the annealed regions, the NAT pairs are thermodynamically stable in vivo. For the organisms with annotated reference genomes, the NATs could be classified into cis- or trans-NATs according to their genomic origins. A pair of cis-NATs is formed by two transcripts derived from two overlapping genomic loci on the opposite strands, while a trans-NAT pair is constituted by the complementary transcripts encoded by two distant genomic loci. Thus, one of the distinguishable features between cis- and trans-NAT pairs is that the complementary regions of the cis-NAT pairs are perfectly annealed while the trans-NAT pairs usually have mis- matches within their annealed regions. However, one common feature is shared by the two NAT categories that the annealed regions should be long and stable enough to ensure the correct formation of the transcript pairs. To date, many organisms do not have their reference genomes, resulting in a big obstacle for NAT identification and classification. i Ten years ago, Zhu’s lab reported that through 24- and 21-nt nat-siRNA-mediated target cleavages, a cis-NAT pair could modulate salt tolerance in Arabidopsis (Arabidopsis thaliana)1. During the past few years, grow- ing evidences pointed to the functional involvement of NATs and nat-siRNAs in diverse biological processes. In rice (Oryza sativa), Jabnoune and his colleagues identified a cis-NAT pair constituted by PHOSPHATE1;2 1College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, PR China. 2Zhejiang Provincial Key Laboratory for Genetic Improvement and Quality Control of Medicinal Plants, Hangzhou Normal University, Hangzhou 310036, China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Y.M. (email: mengyijun@zju.edu.cn) or H.W. (email: whz62@163.com) Scientific Reports \| 6:21666 \| DOI: 10.1038/srep21666 1 Scientific Reports \| 6:21666 \| DOI: 10.1038/srep21666 An integrative pipeline for NAT and phased nat-siRNA discovery And, it will be a hard task for us to obtain evidences to support the scattered siRNA loci without any regular distribution patterns on the NATs. The pioneering work by Zhu’s lab attracted our attention that at least a portion of nat-siRNAs was distributed with defined phases1. Our previous work on Arabidopsis and rice also revealed several pairs of cis-NATs encoding phased nat-siRNAs13. Consistently, several recent works also unveiled many phase-distributed nat-siRNA loci in plants16,31. In this consideration, we developed a program to search for phased nat-siRNAs within the perfectly annealed regions of the NATs, which was integrated into NATpipe (Fig. 1B). If the users have sRNA high-throughput sequencing (HTS) data, NATpipe could be continued to identify the phased nat-siRNA loci. First, the NAT pairs with perfectly annealed regions exceeding a predefined length (an adjustable parameter; default: >80 bp) will be extracted. Then, the sRNAs from HTS data sets (see user manual in Data S1 for the required format of the HTS data) will be mapped onto these NATs. Bowtie (see user manual in Data S1 for parameter setting) is employed for the strand-specific mapping, and the perfectly aligned sRNAs will be retained to search for the phased nat-siRNAs. Based on the previous reports, the annealed regions of the NAT pairs are processed by DCL (Dicer-like) proteins1,16–19, resulting in the production of phased sRNA duplexes with 2-nt 3′ overhangs. This biogenesis pathway is similar to that of the trans-acting siRNAs encoded by the TAS genes32. In this regard, NATpipe recruited a Perl script to search for phased sRNA duplexes with 2-nt 3′ overhangs within the perfectly annealed regions based on the sRNA mapping result (Fig. 1B). The number of the consecutively distributed sRNA duplexes is another adjustable parameter (default: > = 4). Notably, both the sRNAs displaying spatio-temporal expression patterns and the weakly expressed ones might be rarely detectable due to the limited HTS data utilized for this study. Thus, it might be excessively demanded to set a criterion that both strands of each phased sRNA duplex should be evidenced by sRNA HTS data. Instead, we set a more flexible criterion that sequencing evidences should be obtained for either strand of each phased sRNA duplex, which enabled users to identify much more phased nat-siRNA candidates. Scientific Reports \| 6:21666 \| DOI: 10.1038/srep21666 2 www.nature.com/scientificreports/ Figure 1. Summarized workflow of NATpipe. NATpipe utility: a case study on the non-model plant Dendrobium officinale NATpipe utility: a case study on the non model plant Dendrobium officinale To verify the utility of NATpipe for NAT and nat-siRNA discovery, we performed a case study by using RNA sequencing (RNA-seq) data of Dendrobium officinale, a non-model plant species. In our recent study, eight RNA-seq data sets [two biological replicates for each of four organs including root, stem, leaf and flower; NCBI SRA (http://www.ncbi.nlm.nih.gov/sra) accession IDs: SRR2014227, SRR2014230, SRR2014236, SRR2014246, SRR2014297, SRR2014325, SRR2014396 and SRR2014476] were generated, providing 445,430,002 valid reads35. Although the draft genome of Dendrobium officinale has been reported36, the genome assembly and the gene annotations are still far from a satisfied status. Thus, we previously took a de novo strategy for Dendrobium tran- scriptome assembly by utilizing the eight RNA-seq data sets. As a result, 536,558 transcripts ranging from 201 to 21,555 nt were obtained35. In the present study, the 536,558 transcripts were treated as input data for NAT prediction by using NATpipe. As a result, a total of 2,651,469 transcript pairs containing highly complementary regions were identified from the Dendrobium transcriptome based on the BLAST results. These complementary pairs were further classified into 1,269,633 “HC” and 1,741,803 “100-nt” pairs. Then, RNAplex was employed for secondary structure prediction to investigate the annealing potential of the BLAST-predicted transcript pairs. The results of RNAplex were parsed and made a comparison with those of BLAST, and the transcript pairs fulfilling the criteria proposed above were retained as the NAT candidates. As a result, a total of 636,074 NAT pairs were retained, and 436 were identified as cis-NAT pairs considering the single perfectly annealed region for each pair. i p g g p y g p Next, we searched for the phased nat-siRNAs by utilizing eight sRNA HTS data sets (two biological replicates for each of four organs including root, stem, leaf and flower; NCBI SRA accession IDs: SRR2014142, SRR2014143, SRR2014477, SRR2014478, SRR2014146, SRR2014147, SRR2014148 and SRR2014149) reported in our recent study35. To do this, the NATs containing perfectly annealed regions longer than 80 bp were subjected to sRNA mapping. A total of 193,167 annealed regions assigned to 163,654 NAT pairs were included in this analysis. Based on the mapping results, NATpipe searched for the sRNA clusters constituted by four or more phased nat-siRNA candidates. An integrative pipeline for NAT and phased nat-siRNA discovery (A) Two functional modules “NAT prediction” and “Search for phased sRNAs” were integrated into the pipeline. The first module requires de novo assembled transcripts as the input, and the second module requires small RNA (sRNA) HTS data (at least) and degradome HTS data (would be best if available). The parameters in blue color are adjustable. (B) Illustration of the phase-distribute sRNAs identified within the perfectly annealed regions (> 80 bp) of a NAT pair. As an example, eight sRNAs with consistent sequence length were assigned to four phases (four sRNAs on each strand). Each phased sRNA duplex (sRNA1/sRNA8, sRNA2/sRNA7, sRNA3/sRNA6 and sRNA4/sRNA5) possesses 2-nt overhangs at thei 3′ ends (dashed boxes just indicate two 2-nt overhangs for example). Figure 1. Summarized workflow of NATpipe. (A) Two functional modules “NAT prediction” and “Search for phased sRNAs” were integrated into the pipeline. The first module requires de novo assembled transcripts as the input, and the second module requires small RNA (sRNA) HTS data (at least) and degradome HTS data (would be best if available). The parameters in blue color are adjustable. (B) Illustration of the phase-distributed sRNAs identified within the perfectly annealed regions (> 80 bp) of a NAT pair. As an example, eight sRNAs with consistent sequence length were assigned to four phases (four sRNAs on each strand). Each phased sRNA duplex (sRNA1/sRNA8, sRNA2/sRNA7, sRNA3/sRNA6 and sRNA4/sRNA5) possesses 2-nt overhangs at their 3′ ends (dashed boxes just indicate two 2-nt overhangs for example). Degradome sequencing (degradome-seq) is a high-throughput strategy for detection of the degraded RNA intermediates. It is worth mentioning that the degradome-seq libraries include poly(A)-tailed remnants gen- erated during DCL-mediated processing of the sRNA precusors33,34. Thus, we could find evidences from the degradome-seq data to support the processing of the phased nat-siRNA duplexes by DCLs. If users have degradome-seq data (see user manual in Data S1 for the required format of the HTS data), NATpipe could per- form strand-specific mapping of the degradome signatures onto the NATs with phased nat-siRNA loci. Only the perfectly matched signatures will be retained. The degradome signatures with their 5′ ends mapped to the 5′ ends of the phased nat-siRNAs, or mapped to the nucleotides next to the 3′ ends of the last phased nat-siRNAs will be regarded as the evidences supporting specific nat-siRNA loci. An integrative pipeline for NAT and phased nat-siRNA discovery However, one be noticed that most of the Scientific Reports \| 6:21666 \| DOI: 10.1038/srep21666 3 www.nature.com/scientificreports/ degradome-seq libraries were prepared from poly(A)-tailed RNAs, which could not be utilized for detecting the processing signals from the non-Pol II-transcribed transcripts. g g p NATpipe, developed by the Perl language, is compatible for both Windows and Linux operation systems. It is reely available at www.bioinfolab.cn/NATpipe/NATpipe.zip. NATpipe utility: a case study on the non-model plant Dendrobium officinale As a result, 36,033 phased nat-siRNAs assigned to 8,499 clusters were identified within 1,191 annealed regions of 1,034 NAT pairs.hl g p Then, four degradome-seq data sets (four organs including root, stem, leaf and flower; NCBI SRA accession IDs: SRR2012529, SRR2012531, SRR2012580 and SRR2012592) were used to find evidences supporting the pro- cessing of the nat-siRNAs from the NATs. Degradome signatures were mapped onto the NAT pairs encoding phased nat-siRNAs. Based on the mapping results, NATpipe searched for the degradome signatures with their 5′ ends mapped to the 5′ ends of the nat-siRNA candidates, or mapped to the nucleotides next to the 3′ ends of the last phased nat-siRNAs. As a result, 5,739 nat-siRNA candidates (assigned to 3,770 clusters within 524 annealed regions of 502 NAT pairs) were supported by degradome signatures. Finally, a result summary and a detailed report showing the NAT pairs generating phased nat-siRNAs along with degradome-seq evidences were generated. g An example of output result reporting a NAT pair producing phased nat-siRNAs is shown in Table S1. It provides us with the following information: (1) Based on the RNAplex-predicted annealed region between comp175659_c0_seq1 (from 1,904th to 2,956th nucleotide) and comp168422_c0_seq11 (from 1st to 1,053th nucle- otide), the two transcripts might form a NAT pair. (2) Combinatory use of sRNA-seq data from four organs of Dendrobium enabled us to identify 63 nat-siRNAs assigned to 20 consecutive phases. However, when using sRNA-seq data from a single organ, eight nat-siRNAs assigned to seven phases were identified in roots (high- lighted in gray background in Table S1), and nine nat-siRNAs assigned to seven phases (gray background) and 12 nat-siRNAs assigned to seven phases (gray background) were identified in leaves. (3) Detailed information of the degradome signatures (including IDs, expression levels and positions on the NATs) supporting the pro- cessing of the nat-siRNAs is also provided. Based on Table S1, an intelligible figure could be drawn to display phased nat-siRNAs and degradome signatures with organ-specific patterns (Fig. 2). Intriguingly, 58 out of 63 nat-siRNAs detected from eight sRNA HTS data sets were assigned to comp175659_c0_seq1, forming 18 con- secutive phases. Only five nat-siRNAs were assigned to comp168422_c0_seq11. Similar to our result, a previous study on Arabidopsis and rice reported that the nat-siRNAs frequently distributed with a strand bias within the overlapping regions of the cis-NATs16. Concluding remarks and perspectives g p p Here, we provide researchers with an integrative pipeline for NAT prediction by using de novo assembled tran- scriptomes of the non-model plant species. NATpipe also allows users to search for phased nat-siRNAs depending on the availability of sRNA and degradome sequencing data. The publicly available, widely used tools (BLAST, RNAplex and Bowtie), the adjustable parameters, and the detailed user manual ensure the ease of use of NATpipe. NATpipe was written by Perl language, and was compatible for Windows and Linux operation systems. The phased nat-siRNAs along with the degradome-seq evidences facilitate researchers to design further experiments for functional studies on the NATs and the siRNAs. We acknowledge that the endogenous small interfering RNA (endo-siRNA) system of animal has not been characterized as well as that of plants, and the two systems may be different in some aspects. But, increasing evi- dences demonstrated that in addition to the endo-siRNA pathway previously identified in the nematodes, intra- or inter-molecular interactions of RNA precursors could serve as the substrates of Dicer proteins for endo-siRNA generation in Drosophila melanogaster, mice and human. And, a portion of these endo-siRNAs originates from NATs, which is functionally involved in reproduction and neural development23–30,37,38. Although the levels, Scientific Reports \| 6:21666 \| DOI: 10.1038/srep21666 4 www.nature.com/scientificreports/ Figure 2. Graphic presentation of the exemplified output results of NATpipe. (A) Degradome signatures mapped to the 5′ ends of the phased nat-siRNAs in Dendrobium officinale are expressed by asterisks (blue, brown, green and orange for root, stem, leaf and flower respectively). The degradome signal intensity is shown in the histogram. A total of 20 phases were identified within the perfectly annealed region (marked by a red box) between the two transcripts comp175659_c0_seq1 (annealed from 2429th to 2848th nucleotide) and comp168422_c0_seq11 (from 111th to 530th nucleotide) based on small RNA (sRNA) sequencing data. For each phase on a strand of the annealed region, the presence of a nat-siRNA in a specific organ is expressed by a colored bar (blue, brown, green and orange for root, stem, leaf and flower respectively). Based on the sRNA sequencing data, expression levels of the nat-siRNAs are shown in the histograms in (B) (for the siRNAs on comp175659_c0_seq1) and (C) (for the siRNAs on comp168422_c0_seq11). There are two biological replicates of the sRNA sequencing experiments. Please note, the y axes of the three histograms are measured in RPM (reads per million) with exponential increment. Concluding remarks and perspectives Figure 2. Graphic presentation of the exemplified output results of NATpipe. (A) Degradome signatures mapped to the 5′ ends of the phased nat-siRNAs in Dendrobium officinale are expressed by asterisks (blue, brown, green and orange for root, stem, leaf and flower respectively). The degradome signal intensity is shown in the histogram. A total of 20 phases were identified within the perfectly annealed region (marked by a red box) between the two transcripts comp175659_c0_seq1 (annealed from 2429th to 2848th nucleotide) and comp168422_c0_seq11 (from 111th to 530th nucleotide) based on small RNA (sRNA) sequencing data. For each phase on a strand of the annealed region, the presence of a nat-siRNA in a specific organ is expressed by a colored bar (blue, brown, green and orange for root, stem, leaf and flower respectively). Based on the sRNA sequencing data, expression levels of the nat-siRNAs are shown in the histograms in (B) (for the siRNAs on comp175659_c0_seq1) and (C) (for the siRNAs on comp168422_c0_seq11). There are two biological replicates of the sRNA sequencing experiments. Please note, the y axes of the three histograms are measured in RPM (reads per million) with exponential increment. distribution patterns and prevalence of the nat-siRNAs are better characterized in plants, we still hope that the application of NATpipe may extend to the discovery of NATs and nat-siRNAs in animals.f Next, we will make our efforts to improve the functionality of NATpipe, and the presentation of its outputs. For example, the table-based outputs will be converted to graphic ones, taking Fig. 2 as a reference. Besides, the NAT abundances will be considered for expression level-based investigation of the relationships among NATs, nat-siRNAs and degradome signatures from various organs or growth conditions. Summarily, we hope that NATpipe could serve as a useful tool for NAT prediction, nat-siRNA discovery, and related functional studies. g p 13. Chen, D. et al. 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Acknowledgements g The authors would like to thank for the anonymous reviewers for their critical reviews and constructive comments. This research was funded by the National Natural Science Foundation of China [31070298] and [31571349], Science and technology project of Zhejiang Province [2008C12081], Zhejiang Provincial g The authors would like to thank for the anonymous reviewers for their critical reviews and constructive comments. This research was funded by the National Natural Science Foundation of China [31070298] and [31571349], Science and technology project of Zhejiang Province [2008C12081], Zhejiang Provincial Natural Science Foundation of China [LY15C060006], and Hangzhou Scientific and Technological Program [20150432B02] comments. This research was funded by the National Natural Science Foundation of China [31070298] and [31571349], Science and technology project of Zhejiang Province [2008C12081], Zhejiang Provincial Natural Science Foundation of China [LY15C060006], and Hangzhou Scientific and Technological Program [20150432B02]. and [31571349], Science and technology project of Zhejiang Province [2008C12081], Zhejiang Provincial Natural Science Foundation of China [LY15C060006], and Hangzhou Scientific and Technological Program [20150432B02]. Author Contributions Conceived and designed the experiments: Y.M. and H.W. Performed the experiments: D.Y., Y.M. and Z.Z. Analyzed the data: D.Y., Y.M., Z.Z. and J.X. Contributed reagents/materials/analysis tools: D.Y., Z.Z. and J.X. Wrote the paper: Y.M. and H.W. All authors reviewed and discussed the manuscript. References Prediction of trans-antisense transcripts in Arabidopsis thaliana. 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NATpipe: an integrative pipeline for systematical discovery of natural antisense transcripts (NATs) and phase-distributed nat-siRNAs from de novo assembled transcriptomes. Sci. Rep. 6, 21666; doi: 10.1038/srep21666 (2016). How to cite this article: Yu, D. et al. NATpipe: an integrative pipeline for systematical discovery of natural antisense transcripts (NATs) and phase-distributed nat-siRNAs from de novo assembled transcriptomes. Sci. Rep. 6, 21666; doi: 10.1038/srep21666 (2016). 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. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ 6 Scientific Reports \| 6:21666 \| DOI: 10.1038/srep21666
https://openalex.org/W4393090226	https://www.imrpress.com/journal/FBL/29/3/10.31083/j.fbl2903116/pdf	English	null	Host Cell-dependent Modulatory Role of Ras Homolog Enriched in Brain-Like-1 (RhebL1) Protein in Influenza A/NWS/33 Virus-infected Mammalian Cells	Frontiers in bioscience	2,024	cc-by	5,668	Abstract Background: The Mammalian Target of Rapamycin (mTOR) signaling pathway regulates protein phosphorylation and exerts control over major cellular processes. mTOR is activated by the small G-protein Ras Homolog Enriched in Brain (Rheb), which is encoded by the Rheb1 and Rheb-like-1 (RhebL1) genes. There is currently a paucity of information on the role of RhebL1, and specifically its involvement in viral infection. In the present study we investigated the role of RhebL1 during human influenza A/NWS/33 (NWS/33) (H1N1) virus infection of rhesus monkey-kidney (LLC-MK2) cells and human type II alveolar epithelial (A549) cells. Methods: To assess the efficiency of NWS/33 virus replication, the expression of viral nucleoprotein was examined by indirect immunofluorescence (IIF) and the viral yield by fifty percent tissue culture infectious dose assay. An RNA-mediated RNA interference approach was used to investigate the role of RhebL1 during NWS/33 infection. RhebL1 expression was evaluated by IIF, Western blotting, and enzyme- linked immunosorbent assays. A two-tailed Student’s t-test was applied to evaluate differences between groups. Results: RhebL1 was differentially expressed in the cell models used in this study. Silencing of the RhebL1 gene led to increased NWS/33 virus infection in A549 cells, but not in LLC-MK2 cells. Moreover, the expression of hyperphosphorylated cytokeratin 8, a marker of NWS/33 virus infection efficiency, increased in A549 cells depleted of RhebL1 but remained almost unchanged in LLC-MK2 cells. Conclusions: These are the first results showing involvement of the endogenous RhebL1 protein during viral infection. Our data suggests that RhebL1 exerts a host cell-dependent modulatory role during influenza virus infection. RhebL1 appears to be a restrictive factor against NWS/33 virus replication in A549 cells, but not in LLC-MK2. Keywords: influenza A virus; virus-host interaction; RhebL1 protein; small interfering RNA; phosphorylated keratin 8; restriction factor Keywords: influenza A virus; virus-host interaction; RhebL1 protein; small interfering RNA; phosphorylated keratin Short Communication Host Cell-dependent Modulatory Role of Ras Homolog Enriched in Brain-Like-1 (RhebL1) Protein in Influenza A/NWS/33 Virus-infected Mammalian Cells Short Communication Mirko Buttrini1, Flora De Conto1,* 1Department of Medicine and Surgery, University of Parma, 43126 Parma, Italy Correspondence: flora.deconto@unipr.it (Flora De Conto) Academic Editor: Giuseppe Murdaca Submitted: 24 October 2023 Revised: 23 February 2024 Accepted: 4 March 2024 Published: 20 March 2024 Copyright: © 2024 The Author(s). Published by IMR Press. This is an open access article under the CC BY 4.0 license. Publisher’s Note: IMR Press stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Front. Biosci. (Landmark Ed) 2024; 29(3 https://doi.org/10.31083/j.fbl2903116 Short Communication Host Cell-dependent Modulatory Role of Ras Homolog Enriched in Brain-Like-1 (RhebL1) Protein in Influenza A/NWS/33 Virus-infected Mammalian Cells Front. Biosci. (Landmark Ed) 2024; 29(3): 116 https://doi.org/10.31083/j.fbl2903116 lisher’s Note: IMR Press stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. 1. Introduction are encoded by Rheb [9]. Although the role of Rheb1 has been widely established [8,10,11], little is known regarding the involvement of RhebL1 [12]. Moreover, most previous studies have investigated the transient over-expression of RhebL1, rather than the endogenous protein [13]. Successful virus replication relies on specific strate- gies to gain control of key host cell signaling pathways. The Mammalian Target of Rapamycin (mTOR) is a highly con- served signaling pathway that plays a central role in consol- idating the cellular physiology of all eukaryotes. It achieves this by controlling major processes such as protein synthe- sis, cellular growth/proliferation, and apoptosis [1–4]. Hy- peractivated mTOR signaling has been associated with can- cer, diabetes, and the aging process [5–7]. The mTOR pathway plays an important role in viral replication by regulating apoptosis, cell survival, and both transcription and translation mechanisms [14,15]. Specif- ically, the 1918 pandemic strain of influenza A virus re- quires mTORC1 activity during the early replication phases [16]. Moreover, influenza A virus modulates the levels of mTOR RNA and protein [17]. mTOR constitutes the catalytic subunit of two dis- tinct complexes known as mTOR complex 1 (mTORC1) and mTORC2. These have different substrates and func- tions [7]. Specifically, mTORC1 controls the balance be- tween anabolism and catabolism, while mTORC2 governs cytoskeletal behavior and activates various survival and proliferation pathways [3]. Both complexes integrate up- stream environmental information to gate their activation. mTORC1 is activated by the small G-protein Ras Homolog Enriched in Brain (Rheb) in its GTP-bound state, which is promoted by growth factors [8]. The Ras Homolog En- riched in Brain 1 (Rheb1) and Rheb-like-1 (RhebL1) genes Kim et al. [18] studied the involvement of RhebL1 in keratin 8 phosphorylation and reorganization in a human type II alveolar epithelial cell line (A549). In this regard, we previously reported that influenza A/NWS/33 (NWS/33) virus induces keratin 8 hyperphosphorylation in A549 cells, thereby enhancing its replicative efficiency [19]. The rhesus monkey-kidney (LLC-MK2) (simian) and A549 (human) mammalian cell models were previously re- ported to show different levels of permissiveness to in- fluenza A virus infection [19,20]. The aim of the present BSA in PBS. The cells were mounted in buffered glycerol solution (BioMérieux Italia S.p.A, Florence, Italy) and ex- amined through an epifluorescence microscope (Leica Mi- crosystems Srl, Milan, Italy). study was therefore to compare the involvement of RhebL1 protein during NWS/33 virus infection in these two cell models. 2.2 Viral Infection Human influenza A/NWS/33 virus (H1N1; ATCC VR 219) was propagated as described previously [21]. In brief, LLC-MK2 and A549 cells were grown to confluence in shell vials or 6-well plates and then infected at a multiplic- ity of infection (MOI) of 0.1 and one plaque-forming unit (PFU)/cell, respectively. After adsorption for 75 min at 4 °C, the viral inoculum was removed and the cells washed twice with serum-free medium before incubation for the time indicated. 2.4 Western Blotting (WB) Assays WB assays were performed as previously described [22] on cell lysates collected from two replicate wells for each experimental condition. Mouse monoclonal anti-beta- actin IgG (1:400; Santa Cruz Biotechnology) and rabbit polyclonal anti-Rheb2-N-terminal (1:300; Prodotti Gianni, Milan, Italy) antibodies were used for the WB assays. Bound antibodies were detected by anti-mouse (1:5000; Sigma-Aldrich) and anti-rabbit (1:5000; Sigma-Aldrich) IgG alkaline phosphatase-conjugated antibodies. 2. Materials and Methods 2.1 Cells For each cell monolayer, 10 arbitrarily selected fields were analyzed and viral NP-positive cells were expressed as the mean percentage value of the total cell number per field, as estimated by chromatin staining with 4′,6-diamidino-2- phenyl-indole dihydrochloride (DAPI; 2.5 µg/mL; Sigma- Aldrich, Milan, Italy). A549 (TCL 101), LLC-MK2 (BS CL 57), and Madin- Darby canine-kidney (MDCK, BS CL 64) cells were obtained from the Lombardy and Emilia Romagna Ex- perimental Zootechnic Institute (IZSLER, Brescia, Italy). Cells were cultured in either Ham’s F-12 Nutrient Mix- ture (A549) or Earle’s Modified Eagle’s Medium (LLC- MK2 and MDCK) containing 2 mM L-glutamine, 10% fe- tal bovine serum, and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin). All cell lines were validated at IZSLER by Short Tandem Repeat-DNA typing and tested negative for mycoplasma, as per international guidelines. Cells were cultured in a humidified incubator at 37 °C and 5% CO2, and culture reagents were purchased from Euro- Clone (Milan, Italy). 2.3 Indirect Immunofluorescence (IIF) Assays Monolayers of LLC-MK2 and A549 cells were fixed and permeabilized in methanol for 5 min at –20 °C. The cells were then washed with phosphate-buffered saline (PBS, pH 7.4; 7 mM Na2HPO4, 1.5 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl), blocked with 1% bovine serum albu- min (BSA; PAA Laboratories GmbH, Cölbe, Germany) in PBS, and incubated for 1 h at 37 °C with primary antibodies diluted in 0.2% BSA in PBS. After three washes with PBS, the cells were incubated for 45 min at 37 °C with the sec- ondary antibodies diluted in 0.2% BSA in PBS. For nega- tive controls, the primary antibodies were replaced by 0.2% 2.5 RNA-mediated RNA Interference To obtain transient knockdown of the RhebL1 gene, we used siRNA (5′-GCAGGAUGAGUACAGCAUU-3′; 3′-CGUCCUACUCAUGUCGUAA-5′) and universal neg- ative siRNA as a negative control as previously described [18]. To avoid toxicity effects induced by gene silenc- ing, preliminary assays (data not shown) were performed to evaluate the cellular viability with trypan blue exclu- sion assays [19]. LLC-MK2 and A549 cells at 75% con- fluence in shell vials or 6-well plates were transfected for 48 h with RhebL1 siRNA (15 nM) or control siRNA (non- effective scrambled double-stranded siRNA) using MIS- SION® siRNA transfection reagent. All reagents for RNA- mediated RNA interference were purchased from Sigma- Aldrich. 1. Introduction To achieve this, we evaluated the effect of small in- terfering RNA (siRNA)-mediated silencing of the RhebL1 gene on both viral replication efficiency and on the phos- phorylation of keratin 8. For IIF assays, mouse monoclonal anti-influenza A virus nucleoprotein (NP) (1:30, BioMérieux), goat poly- clonal anti-RhebL1 (1:15; Santa Cruz Biotechnology, Bergheimer, Germany), and rabbit anti-phosphorylated ker- atin 8 on serine 431 (1:100, ThermoFisher Scientific, Waltham, MA, USA) antibodies were used as described previously [19]. These antibodies were in turn detected by Alexa Fluor 568 goat anti-mouse IgG (1:500; Molec- ular Probes, Eugene, OR, USA), Alexa Fluor 467 donkey anti-goat IgG (1:500; Molecular Probes), and fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG (1:70; Li StarFish, Milan, Italy) antibodies, respectively. We found that RhebL1 was expressed at a higher level in A549 cells than in LLC-MK2 cells and has a host cell- dependent modulatory role in NWS/33 replication. More specifically, RhebL1 acts as a restriction factor against NWS/33 virus replication and keratin 8 hyperphosphory- lation in A549 cells, but did not to appear to have an active modulatory role in LLC-MK2 cells. 2.6 Fifty Percent Tissue Culture Infectious Dose (TCID50) Assay The viral yields in culture supernatants from MDCK cells were assessed as previously reported [23]. 2 Fig. 1. Analysis of Ras Homolog Enriched in Brain Like-1 (RhebL1) expression in uninfected and human influenza A/NWS/33 (NWS/33)-infected rhesus monkey-kidney (LLC-MK2) and A549 cells. RhebL1 protein expression in uninfected LLC-MK2 cells (A) and A549 cells (B) was investigated using indirect immunofluorescence (IIF) assays. Images were recorded with a conventional fluorescence microscope. Scale bar = 20 µm. (A,B) The insets shown at the bottom of each figure represent the higher magnification (2000×) of the cell/s included within the frame shown in the microscopic field. (C) Quantitative evaluation of RhebL1 in uninfected and NWS/33-infected (multiplicity of infection (MOI) = 1 PFU/cell, 24 h infection) LLC-MK2 cells and A549 cells, as determined by Enzyme-linked Immunosorbent Assay (ELISA). Values represent the mean of three independent experiments. Error bars in the graphs represent the standard deviation. p = 0.037. Fig. 1. Analysis of Ras Homolog Enriched in Brain Like-1 (RhebL1) expression in uninfected and human influenza A/NWS/33 (NWS/33)-infected rhesus monkey-kidney (LLC-MK2) and A549 cells. RhebL1 protein expression in uninfected LLC-MK2 cells (A) and A549 cells (B) was investigated using indirect immunofluorescence (IIF) assays. Images were recorded with a conventional fluorescence microscope. Scale bar = 20 µm. (A,B) The insets shown at the bottom of each figure represent the higher magnification (2000×) of the cell/s included within the frame shown in the microscopic field. (C) Quantitative evaluation of RhebL1 in uninfected and NWS/33-infected (multiplicity of infection (MOI) = 1 PFU/cell, 24 h infection) LLC-MK2 cells and A549 cells, as determined by Enzyme-linked Immunosorbent Assay (ELISA). Values represent the mean of three independent experiments. Error bars in the graphs represent the standard deviation. * p = 0.037. 2.7 Enzyme-linked Immunosorbent Assay (ELISA) lution was added to each well before sealing the plate and incubating for 30 minutes at 37 °C. In the last step, the plate was washed five times and 90 µL of tetramethylbenzidine substrate solution was then added to each well. The plate was once again sealed and incubated for 15 minutes at 37 °C. Finally, 50 µL of stop solution was added to each well and the optical density (OD) absorbance at 450 nm was im- mediately read. To quantify RhebL1 protein, the human RHEBL1 (GTPase RhebL1) ELISA kit was employed according to the manufacturer’s guidelines (Fine Biotech Co., Wuhan, Hubei, China). First, a standard curve was created using a standard control and starting at a protein concentration of 1000 pg/mL, followed by 2-fold serial dilutions from 1/2 to 1/64. The RhebL1 concentration in samples was calculated based on the standard curve. Briefly, 100 µL of sample (or prepared standard points) was added to each dedicated well of a 96-well plate. This was sealed and incubated for 90 minutes at 37 °C, after which the plate was washed twice and 100 µL of biotin-labeled antibody working solution was added to each well. The plate was then sealed and incubated again for 60 minutes at 37 °C. After three washes, 100 µL of horseradish peroxidase-streptavidin conjugate working so- Regarding the OD calculation, the relative OD ab- sorbance at 450 nm was obtained with the following for- mula: relative OD450 = (OD450 of each well) – (OD450 of blank well) The standard curve was plotted as the relative OD450 of each standard point (Y-axis) versus each respective con- centration (X-axis). The RhebL1 concentration of each sample was interpolated from the standard curve. 3 Fig. 2. RhebL1 protein restricts productive NWS/33 virus infection in A549 cells. (A–C) LLC-MK2 and A549 cells were treated for 48 h with either control siRNA or RNA-mediated interference against RhebL1. (A) RhebL1 expression in LLC-MK2 and A549 cells was evaluated by Western blotting (WB), with beta-actin used as the protein loading control. (B) Histogram showing the percentage of viral NP-positive LLC-MK2 and A549 cells following treatment for 48 h with either control siRNA or RhebL1 siRNA, as evaluated by IIF after infection with NWS/33 virus (MOI = 0.1 PFU/cell, 24 h). 2.8 Statistical Analysis ELISA was used to determine the concentration of RhebL1 in cellular homogenates of either uninfected or NWS/33-infected (MOI = 1 PFU/cell, 24 h) LLC-MK2 and A549 cells (Fig. 1C). The results showed much higher ex- pression of RhebL1 in uninfected A549 cells than in unin- fected LLC-MK2 cells. NWS/33 virus infection did not al- ter the RhebL1 concentration in LLC-MK2 cells, but caused a significant decrease in A549 cells. GraphPad Prism software (Version 10.0.0 (153), GraphPad Software LLC, Boston, MA, USA) was em- ployed for the statistical analysis. A two-tailed Student’s t-test was used to evaluate differences between cells under study. p-values < 0.05 were considered statistically signif- icant. 2.7 Enzyme-linked Immunosorbent Assay (ELISA) (C) The viral yields in the supernatants of LLC-MK2 and A549 cells were evaluated by the fifty percent tissue culture infectious dose (TCID50) assay in MDCK cells. Values represent the mean of two independent experiments. Error bars represent the standard deviation. * p = 0.0056 in (B) and * p = 0.0458 in (C). n.s. p = 0.189 in (B) and n.s. p = 0.4198 in (C). n.s., not statistically significant. Fig. 2. RhebL1 protein restricts productive NWS/33 virus infection in A549 cells. (A–C) LLC-MK2 and A549 rotein restricts productive NWS/33 virus infection in A549 cells. (A–C) LLC-MK2 and A549 cells were treated Fig. 2. RhebL1 protein restricts productive NWS/33 virus infection in A549 cells. (A–C) LLC-MK2 and A549 cells were treated for 48 h with either control siRNA or RNA-mediated interference against RhebL1. (A) RhebL1 expression in LLC-MK2 and A549 cells was evaluated by Western blotting (WB), with beta-actin used as the protein loading control. (B) Histogram showing the percentage of viral NP-positive LLC-MK2 and A549 cells following treatment for 48 h with either control siRNA or RhebL1 siRNA, as evaluated by IIF after infection with NWS/33 virus (MOI = 0.1 PFU/cell, 24 h). (C) The viral yields in the supernatants of LLC-MK2 and A549 cells were evaluated by the fifty percent tissue culture infectious dose (TCID50) assay in MDCK cells. Values represent the mean of two independent experiments. Error bars represent the standard deviation. * p = 0.0056 in (B) and * p = 0.0458 in (C). n.s. p = 0.189 in (B) and n.s. p = 0.4198 in (C). n.s., not statistically significant. 3.2 RhebL1 Gene Silencing Increases Influenza A/NWS/33 Virus Replication in A549 Cells but not in LLC-MK2 Cells 3.1 Differential RhebL1 Expression in Uninfected and Influenza A/NWS/33 Virus-infected LLC-MK2 and A549 Cells Next, we used an RNA-mediated RNA interference approach to study the effect of RhebL1 depletion on NWS/33 virus infection in LLC-MK2 and A549 cells, as described in the Methods section. The effectiveness of RhebL1 depletion in both models was first assessed by WB (Fig. 2A). The expression and cellular distribution of endoge- nous RhebL1 protein was first examined with the IIF as- say in LLC-MK2 and A549 cells (Fig. 1A,B). RhebL1 ex- pression was lower in uninfected LLC-MK2 cells than un- infected A549 cells. Furthermore, RhebL1 showed diffuse staining with prevalent accumulation in the perinuclear re- gion (see insets in Fig. 1A,B). Subsequently, the control cells and cells depleted of RhebL1 were infected with NWS/33 virus (MOI = 0.1 PFU/cell, 24 h) prior to the IIF assay. Compared to con- 4 Fig. 3. RhebL1 protein restricts the expression of phosphorylated keratin 8 in A549 cells. LLC-MK2 and A549 cells were treated for 48 h with control siRNA (A,C) or with RNA-mediated interference (B,D). The expression of phosphorylated keratin 8 in LLC-MK2 cells (A,B) and in A549 cells (C,D) was then visualized by IIF. Images were recorded using a conventional fluorescence microscope. Scale bar = 20 µm. Fig. 3. RhebL1 protein restricts the expression of phosphorylated keratin 8 in A549 cells. LLC-MK2 and A549 cells were treated for 48 h with control siRNA (A,C) or with RNA-mediated interference (B,D). The expression of phosphorylated keratin 8 in LLC-MK2 cells (A,B) and in A549 cells (C,D) was then visualized by IIF. Images were recorded using a conventional fluorescence microscope. Scale bar = 20 µm. expression and perinuclear accumulation of phosphorylated keratin 8 were observed in A549 cell monolayers (see ar- rows in Fig. 3D). trol cells, the percentage of viral NP-positive cells increased significantly in A549 cells depleted of RhebL1, as shown in Fig. 2B. Conversely, depletion of RhebL1 in LLC-MK2 cells had no effect, or only a slightly negative effect on NWS/33 virus infection. Using the TCID50 assay, we also investigated the effect of RhebL1 depletion on the emer- gence of viral progeny. As shown in Fig. 2C, the results were consistent with those of IIF. 4. Discussion Viruses are master manipulators of cell functions and can hijack key signaling pathways to modulate cell survival and ensure their replication. Since the mTOR signaling pathway is located at the crossroads of several fundamen- tal cellular pathways, many viruses have developed spe- cific mechanisms that target this major biological switch, thereby inducing metabolic reprogramming [24,25]. 3.3 RhebL1 Gene Silencing Increases the Expression of Phosphorylated Keratin 8 in A549 Cells We previously reported that NWS/33 virus infection can stimulate hyperphosphorylation of keratin 8 on serine 431, thereby promoting virus replication in A549 cells but not in LLC-MK2 cells [19]. In view of the involvement of RhebL1 in keratin 8 phosphorylation in A549 cells [18], we next evaluated the effect of RhebL1 gene silencing on the expression of phosphorylated keratin 8 in both cell models. The focus of the present study was on RhebL1, one of the modulators of the mTOR pathway, as to our knowl- edge there is no data regarding the involvement of this pro- tein in viral infection. Several lines of evidence have impli- cated the mTOR pathway in viral infection. The influenza A virus is thought to induce autophagy by overwhelming the mTOR pathway, thereby leading to its successful repli- cation [26–28]. Furthermore, the respiratory syncytial virus induces autophagy and suppresses the mTOR pathway to To this end, control cells and cells depleted of RhebL1 were examined by IIF (Fig. 3A–D). No apparent changes in the expression of phosphorylated keratin 8 were observed in LLC-MK2 cells (see Fig. 3B vs. 3A). In contrast, increased 5 the host biosynthetic machinery, while favoring viral tran- scription and protein synthesis. This concurs with previ- ous data showing that mTOR suppression during PR8 in- fluenza virus infection increased viral transcription [42]. Ranadheera et al. [16] also observed downregulation of the mTOR pathway at early time points during influenza H1N1 (1918 strain) virus infection. However, this reverted to a steady state level at later post-infection time points, allow- ing the production of viral progeny. evade the host’s immune defenses, thus favoring its survival within the host [29]. Arunachalam et al. [30] also reported the mTOR pathway was downregulated in the plasmacytoid cells of subjects with Coronavirus disease 2019 (COVID- 19). Conversely, Appelberg et al. [31] showed that replica- tion of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) increased mTOR signaling, thereby en- suring the translation of viral proteins and allowing viral assembly [32]. With regard to the modulatory effects of RhebL1 and the possible impacts on virus infection, Yuan et al. [34] also reported a relationship with NF-kappa B-mediated gene transcription. Downregulation of RhebL1 induced by ei- ther viral infection or gene silencing could repress cellular antiviral responses triggered by NF-kappa B, as previously observed during influenza virus infection [43,44]. Availability of Data and Materials The datasets used and/or analyzed during the current study are available from the corresponding author upon rea- sonable request. 3.3 RhebL1 Gene Silencing Increases the Expression of Phosphorylated Keratin 8 in A549 Cells The aim of this study was to evaluate the involve- ment of the RhebL1 protein, an activator of the mTOR path- way, during NWS/33 virus infection in two mammalian cell models with different levels of permissivity [19,20]. RhebL1 is highly expressed in cells of the nervous sys- tem [33] and in other tissues [34], suggesting it has essen- tial regulatory roles in a variety of cell types. The present study found a much higher level of RhebL1 expression in human A549 cells than in monkey LLC-MK2 cells. Given this differential expression, it is conceivable that RhebL1 performs different types of regulatory functions in these two cell models. 5. Conclusions Understanding the effects of host cell metabolic sta- tus on virus replication helps to identify mediators of viral tropism and to explain why some cells are more prone to virus infection than others. This is the first report on the host cell-dependent regulatory role of RhebL1 protein dur- ing influenza A virus infection. Our study has revealed new information on the complex metabolic landscape of cells in- fected with the influenza virus. This is worthy of further in- vestigation with a view to developing broad-range antiviral therapies. Specifically, RhebL1 has been shown to interact with molecules that activate signal transduction pathways for different protein kinases, including the phosphorylation of keratin [13,18,35–38]. Similar to the effect of chemical activators of phosphorylation [19], RhebL1 gene silencing was shown to significantly increase the replication cycle of NWS/33 virus and the phosphorylation of keratin 8 in A549 cells, contrary to what is observed in LLC-MK2 cells. Keratin 8 phosphorylation plays a significant role in regulating keratin filament organization, associations with binding proteins, and modulation of the cell cycle. Previ- ous studies have emphasized the role of phosphorylation in viral pathogenesis. In this regard, Padilla-Mendoza et al. [39] focused on the expression of phosphorylated proteins, including keratin 8, and their association with the progres- sion of cervical lesions caused by papillomavirus. Toivola et al. [40] demonstrated that site-specific keratin 8 phos- phorylation is a marker of the progression of liver diseases caused by hepatitis C virus. We previously reported that increased keratin 8 phosphorylation on serine 431 favored virus replication during influenza A virus infection of A549 cells [19]. Abbreviations A549, human type II alveolar epithelial; ACRYL, acrylamide; BSA, bovine serum albumin; DAPI, 4′,6- diamidino-2-phenylindole dihydrochloride; IIF, indirect immunofluorescence; LLC-MK2, rhesus monkey-kidney; MDCK, Madin-Darby canine-kidney; MOI, the multi- plicity of infection; mTOR, Mammalian Target of Ra- pamycin; NP, nucleoprotein; n.s., not statistically signif- icant; NWS/33 virus, human influenza A/NWS/33 virus; PBS, phosphate-buffered saline; PFU, plaque-forming units; p.i., post-infection; Rheb1, Ras Homolog Enriched in Brain 1; RhebL1, Ras Homolog Enriched in Brain Like- 1; siRNA, small interfering RNA; TCID50, fifty % tissue culture infectious dose; WB, Western blotting. RhebL1 was recently found to induce robust phos- phorylation of interferon regulatory factor 3, thereby act- ing as a trigger of innate immunity [41]. In this regard, our data indicates that RhebL1 has a negative regulatory role on NWS/33 virus infection in permissive A549 cells. Accord- ingly, we observed a significant decrease in virus-induced expression of RhebL1 at 24 h after NWS/33 virus infec- tion in A549 cells, but not in LLC-MK2 cells. Moreover, RhebL1 gene silencing stimulated NWS/33 virus replica- tion in A549 cells, but not in LLC-MK2 cells. Hence, we speculate that downregulation of RhebL1 in A549 cells might help the NWS/33 virus to decrease the activity of References [1] Fingar DC, Blenis J. Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression. Oncogene. 2004; 23: 3151–3171. [22] De Conto F, Fazzi A, Razin SV, Arcangeletti MC, Medici MC, Belletti S, et al. Mammalian Diaphanous-related formin-1 re- stricts early phases of influenza A/NWS/33 virus (H1N1) in- fection in LLC-MK2 cells by affecting cytoskeleton dynamics. Molecular and Cellular Biochemistry. 2018; 437: 185–201. [2] Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes & Development. 2004; 18: 1926–1945. [3] Liu GY, Sabatini DM. mTOR at the nexus of nutrition, growth, ageing and disease. Nature Reviews. Molecular Cell Biology. 2020; 21: 183–203. Molecular and Cellular Biochemistry. 2018; 437: 185–201. [23] De Conto F, Di Lonardo E, Arcangeletti MC, Chezzi C, Medici MC, Calderaro A. Highly dynamic microtubules improve the ef- fectiveness of early stages of human influenza A/NWS/33 virus infection in LLC-MK2 cells. PloS One. 2012; 7: e41207. [4] Marques-Ramos A, Cervantes R. Expression of mTOR in nor- mal and pathological conditions. Molecular Cancer. 2023; 22: 112. infection in LLC-MK2 cells. PloS One. 2012; 7: e41207. [24] Pant A, Dsouza L, Yang Z. Alteration in Cellular Signaling and Metabolic Reprogramming during Viral Infection. mBio. 2021; 12: e0063521. [5] Lu ZH, Shvartsman MB, Lee AY, Shao JM, Murray MM, Klad- ney RD, et al. Mammalian target of rapamycin activator RHEB is frequently overexpressed in human carcinomas and is criti- cal and sufficient for skin epithelial carcinogenesis. Cancer Re- search. 2010; 70: 3287–3298. [25] Dunn EF, Connor JH. HijAkt: The PI3K/Akt pathway in virus replication and pathogenesis. Progress in Molecular Biology and Translational Science. 2012; 106: 223–250. [6] Oh WJ, Jacinto E. mTOR complex 2 signaling and functions. Cell Cycle (Georgetown, Tex.). 2011; 10: 2305–2316. [26] Ma J, Sun Q, Mi R, Zhang H. Avian influenza A virus H5N1 causes autophagy-mediated cell death through suppression of mTOR signaling. Journal of Genetics and Genomics = Yi Chuan Xue Bao. 2011; 38: 533–537. [7] Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012; 149: 274–293. [8] Long X, Lin Y, Ortiz-Vega S, Yonezawa K, Avruch J. Rheb binds and regulates the mTOR kinase. Current Biology: CB. 2005; 15: 702–713. [27] Zhang RH, Zhang HL, Li PY, Li CH, Gao JP, Li J, et al. Author Contributions FDC designed the research study. MB and FDC per- formed the research. MB and FDC analyzed the data. MB and FDC wrote the manuscript. Both authors contributed to editorial changes in the manuscript. Both authors read and 6 PI3K-Akt-mTOR signalling pathway. Nature Reviews. Micro- biology. 2008; 6: 266–275. PI3K-Akt-mTOR signalling pathway. Nature Reviews. Micro- biology. 2008; 6: 266–275. approved the final manuscript. Both authors have partici- pated sufficiently in the work and agreed to be accountable for all aspects of the work. [15] Cooray S. The pivotal role of phosphatidylinositol 3-kinase-Akt signal transduction in virus survival. The Journal of General Vi- rology. 2004; 85: 1065–1076. The authors declare no conflict of interest. [21] Arcangeletti MC, De Conto F, Ferraglia F, Pinardi F, Gatti R, Orlandini G, et al. Host-cell-dependent role of actin cytoskeleton during the replication of a human strain of influenza A virus. Archives of Virology. 2008; 153: 1209–1221. Acknowledgment Not applicable. [17] Sharma S, Chatterjee A, Kumar P, Lal S, Kondabagil K. Upregu- lation of miR-101 during Influenza A Virus Infection Abrogates Viral Life Cycle by Targeting mTOR Pathway. Viruses. 2020; 12: 444. Funding [18] Kim HJ, Byun HJ, Park MK, Kim EJ, Kang GJ, Lee CH. Novel involvement of RhebL1 in sphingosylphosphorylcholine- induced keratin phosphorylation and reorganization: Binding to and activation of AKT1. Oncotarget. 2017; 8: 20851–20864. This work was supported by grants allocated to Flora De Conto by the Italian Ministry of Education, University and Research (MIUR) (“Fondo per il finanziamento delle attività di base di ricerca (FFABR) – 2017”). The founders had no role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript. [19] De Conto F, Conversano F, Razin SV, Belletti S, Arcangeletti MC, Chezzi C, et al. Host-cell dependent role of phosphorylated keratin 8 during influenza A/NWS/33 virus (H1N1) infection in mammalian cells. Virus Research. 2021; 295: 198333. [20] De Conto F, Chezzi C, Fazzi A, Razin SV, Arcangeletti MC, Medici MC, et al. Proteasomes raise the microtubule dynamics in influenza A (H1N1) virus-infected LLC-MK2 cells. Cellular & Molecular Biology Letters. 2015; 20: 840–866. Conflict of Interest The authors declare no conflict of interest. Ethics Approval and Consent to Participate Not applicable. [16] Ranadheera C, Coombs KM, Kobasa D. Comprehending a Killer: The Akt/mTOR Signaling Pathways Are Temporally High-Jacked by the Highly Pathogenic 1918 Influenza Virus. EBioMedicine. 2018; 32: 142–163. 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[40] Toivola DM, Ku NO, Resurreccion EZ, Nelson DR, Wright TL, Omary MB. Keratin 8 and 18 hyperphosphorylation is a marker of progression of human liver disease. Hepatology (Baltimore, Md.). 2004; 40: 459–466. [33] Parmar N, Bonneau A. Production and characterization of a highly specific and potent antibody capable of detecting endoge- nous RhebL1 protein in mammalian tissues and cell lines. Jour- nal of Biotech Research. 2010; 2: 21–33. nal of Biotech Research. 2010; 2: 21–33. [41] Yu JH, Moon EY, Kim J, Koo JH. Identification of Small GT- Pases That Phosphorylate IRF3 through TBK1 Activation Using an Active Mutant Library Screen. Biomolecules & Therapeutics. 2023; 31: 48–58. [34] Yuan J, Shan Y, Chen X, Tang W, Luo K, Ni J, et al. Identifi- cation and characterization of RHEBL1, a novel member of Ras family, which activates transcriptional activities of NF-kappa B. Molecular Biology Reports. 2005; 32: 205–214. [42] Yeganeh B, Ghavami S, Rahim MN, Klonisch T, Halayko AJ, Coombs KM. 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https://openalex.org/W2045352200	https://www.biodiversitylibrary.org/partpdf/68330	English	null	XLIII.—<i>The Nomenclature of European Helices</i>	Annals & magazine of natural history	1,901	public-domain	2,199	* “Guide to the Study of Helices,” Manual of Conchology, (2) ix. (1894). “ ” ( ) + “Ueber einige Nomenclaturfragen,” in Nachrbl. d. D. malak, Gesellsch., December 1900, pp. 161-178. Ann. & Mag. N. Hist. Ser. 7. Vol. viii. On the Nomenclature of Huropean Heli On the Nomenclature of Huropean Heli 325 independence, and after five days from their first exit the parent fish treated them as though the time had come for them to look after themselves; and soon after he took no further trouble with them, except in the way of fighting any supposed enemy that was introduced into the tank. ‘‘T may add that I have since observed other fish, with the same result: I mean, of course, other Chromides.”’ XLITI.—The Nomenclature of Huropean Helices. By Henry A. Piussry, Se.D. PERSONS acquainted with the literature of EKuropean land- snails are aware that during the last century an almost incredible number of generic and subgeneric names were bestowed upon the Helices. Up to this time hardly two authors can be found who use the same set of generic names for these mollusks, save perhaps those who adhere to Lamarckian simplicity. When writing my book upon Helices, I found that there was no standard or established classification or nomenclature ; and while the formulation of a new and, I believe, phylo- genetic classification was what chiefly interested me, I thought it also essential that the nomenclature be placed upon a secure basis. My results, so far as classification is concerned, have met with some approval from those best qualified to judge; but various protests have been made against the changes in generic nomenclature. The appear- ance of a criticism of some seventeen pages’ length, from the pen of the well-known German conchologist Dr. O. von Mél- lendorff f, calls for the reply which I have not felt needful in the case of less conspicuous criticisms. Controversy over names is a notoriously barren employment. Dr. v. Mollendorff rightly reports that in bringing together a number of old sectional and group names under a single generic head, I have selected for such generic name that of the oldest-published group involved, This principle, he goes on to say, is in his opinion to be discarded (1a) if the oldest group-name is based upon an extreme form of the genus, in which case he would select a later name for the genus, and one based upon a species of medium development for the 23 A. On the Nomenclature of Huropean Heli Pilsbry on the 326 group; (10) it is premature to follow the strict rule of priority when the earliest name was based upon a species not positively known to belong to the genus as built up ; (2) the rule of giving page-priority in the case of two or more names for the same group, appearing in one book, he does not recognize, but would select the supposedly most typical from among such names; (3 & 4) after discussing the question of names applied to composite groups, such as those of almost all old authors, Dr. v. Méllendorff states that he considers a generic name preoccupied only when a prior homonym is actually in use. y y To these proposed exceptions to the strict rule of priority I would reply that any exception gives opportunity for those individual differences of opinion which it 1s the sole purpose of rules of nomenclature to obviate. Exception (1a) opens the door to endless discussion as to what is or is net an aberrant species. ‘lo my mind the examples cited by von Mollendorff are not such. Thus, Helicigona lapicida is typical of its genus in soft anatomy, and aberrant in a single character of the shell alone, i.e. the carination. Is a specific character of this sort enough to cause us to disregard the rule of priority? And, then, who is to decide upon what is the natural type of a genus? for here theoretical consider- ations will rule. I grant the force of the objection no. (1 8) ; it applies, however, to no Huropean Helicide. Regarding exception (2) it should be said that if page-priority be rejected there is absolutely no rule to cover cases of two or more names for one group in one book. I therefore hold that, as Jordan says, “in case of twins, primogeniture con- trols.”” Objection no. (4) seems to me to be wholly im- possible of application. How are we to tell whether a name proposed in Coleoptera, for instance, will not become a valid genus, even if now temporarily relegated to synonymy? We are constantly taking up and using names long supposed to be synonyms. ‘The concrete cases discussed by v. Méllen- dorff are as follows :— Of, Man. Conch. ix. p. 258, and Proc. Acad, Nat. Sci. Philad. 1897, p. 359, under H. chionobasis. + The Crustacea of the ‘ Fauna Japonica’ appeared in parts, the first issued in 1833, the last in 1850. Trichia was in “decas quinta 1841” (p. 109). The dates are printed at the foot of the first page of each fascicle. Xerophila, Helicella. This is a case of unusual difficulty, from the fact that all the early authors placed the species of the modern genus in company with many really diverse forms. Whether I took the best course in using the name Helicella remains open to doubt. At all events, the supposed use of that name, prior to Férussac, alleged by von Mollendorff, is easily disposed of. In his ‘ Extrait du Cours de Zoologie,’ 1812, p. 115, Lamarck enumerates the genera of Colimacés—Heélice, Nomenclature of European Helice Ai Heélicelle, Hélicine, Maillot, &c.,—but defines none of them, and gives no Latin names. Blainville, in his ‘Manuel’ of 1825, also mentions f/élicelle, Lam., for Helix obvoluta, but he too fails to Latinize the French vernacular name. This leaves the way clear for Risso’s restriction of Helicella, Fér., in 1826. However, the real point is that we do not know the date of Férussac’s ‘ Prodrome’ with certainty, while I have shown that Gray in 1821 proposed the name Jacosta for a species of the genus . It may well prove that in this case my decision will require revision or reversal; but if so, the name in common use, Xerophila, can in no case be used for the group, as it dates from 1837. Of, Man. Conch. ix. p. 258, and Proc. Acad, Nat. Sci. Philad. 1897, p. 359, under H. chionobasis. p + The Crustacea of the ‘ Fauna Japonica’ appeared in parts, the first issued in 1833, the last in 1850. Trichia was in “decas quinta 1841” (p. 109). The dates are printed at the foot of the first page of each fascicle. Helicodonta, Gonostoma. Any attempt to overthrow Helicodonta in favour of Gono- stuma will be seen to be futile when it is really understood that (1) Helicodonta of Férussac, proposed for Helices with toothed or sinuous mouths, was restricted by Risso, in 1826, to the single species obvoluta; and (2) Gonostoma, Held, 1837, is preoccupied by (Gonostoma, Rafinesque, 1810, a name in common and accepted use by ichthyologists (see Giinther, ‘ The Study of Fishes,’ p. 629, 1880). There isa genus //elicodon in the ‘Tankerville Catalogue, p. 35 (1825), which includes species of Polygyra, Sagda, Anostoma, Cepolis, and Pleurodonte. Hygromia, Fruticicola, Trichia. Dr. von Millendorff agrees with me that Hygromia should stand for the genus commonly known under one or other of the above names; but he prefers Férussac’s form Hygro- manes. As I have elsewhere held, this was intended for a noun in the plural and is not acceptable, besides being a heterogeneous section never yet restricted. I therefore adhere to Risso’s name [ygromia. Regarding the use of the subgeneric term Trichia, I would say that Trichia, de Haan, bears the date of 1841, not 1850, as von Méllendorff seeks to prove t+. ‘This leaves the priority with Trichia, Hartmann, 1840. But it seems to me that Fruticicola of Held, 1857, should have preference on account of its earlier date. Von Mollendorff states that the first species of Fruéicicola was fruticum, Miill., “ Krste Art ist Jruticum, Mill., so dass eigentlich Fruticicola fiir Eulota einzutreten hatte’’; but had the original article by Held in the ‘ Isis’ been consulted, it would have been seen that fruti- cum is not the first but the seventeenth species in Held’s list. As H. fruticum was eliminated from the group by Hartmann three years later, the misgivings expressed by von Mollen- dorff lest I should unsettle the name Hu/ota are groundless. © One of the very few cases where I could not consult an original work, and quoted at second-hand, was in the use of Schliiter’s name Perjoratella. I procured the original work a year or two ago, and find that Westerlund, whom I 328 the Nomenclature of European Helices. followed, was wrong; and the re-arrangement tabulated by von Mollendorff (p. 171) is to be substituted for mine. Campyliea, [Helicigona. It is admitted that my use of Helicigona for the Campylea group of Europe is justified, but von Mollendorff contends that Mérch deserves the credit for such use. This may be true: Mérch was a good and great conchologist in his day and generation, and \| am only sorry that he did not convince every body that they should adopt Helicigona. It is claimed that I erred in putting the carinated H. lapicéda in a section by itself, whereas it should be included with the unkeeled H. cornea. This is a matter of opinion, upon which no one could wish to dogmatize; but one would not suppose that a writer who considers Plectotropis a distinct genus from Agista on account of the keel (there are no anatomical diflerences) would criticize the course I have taken. In conclusion, von Méllendorff suggests that, in pursuance of his rule no. 3, the genus might be called ‘‘ Campylea (Beck) v. Iher.” ‘This course would oppose the rule of priority, and is further open to objection from the fact that v. Ihering included the American Hpiphragmophora species in Campylaa, although I have shown that they are widely different anatomically. Section Otala, Schumacher, 1817. After some discussion of the limits of this group and of my use of the term Otala, Dr. von Méllendorff has cast doubt upon what I expressly and, I think, advisedly stated to be the facts in the case. 1 said (‘ Manual,’ p. 323): “ The name Otala was proposed for three species, placed in two sections. Section a contained hamastoma (which being the Some Questions of Myriopod Nomenclatu 329 type of a prior genus must be eliminated) and afomaria, a new name for lactea, Miill. Section 6 contained the Helix sulcata of Miiller, a form which Swainson, in 1840, made the type of his group Plicadomus. These eliminations leave H., lactea the valid nucleus of Schumacher’s group. .. . Beck’s use of Otala has no bearing upon the case, as he included none of Schumachev’s species in his group.” Against this von Méllendorff quotes from Herrmannsen and Beck, as he had not seen Schumacher’s work ; and after further remarks concludes to use the name Marmorana, Hartmann, for the group, placing “ Otala, Pilsbry,” as a synonym thereto. This conclusion cannot, however, be maintained, since Marmorana was based upon the species serpentina, which belongs not to the group under discussion, but to Lberus. Hartmann’s sole reference to Marmorana is the passage (apropos of a certain malformation) which reads: “ Herr Grat Porro hielt sie fir sehr merkwiirdig und sandte mir mehrere solche von unserer Huparyphia, auch von Marmorana serpentina; wirklich ist die Ursache dieser abnormen Fort- setzung schwierig zu ermitteln,” &c. Thus was Marmorana established. And von Mollendorff gravely concludes: “ Danach wurde Marmorana auf die Formenkreise von vermicu’ata und lactea zu beschriinken sein”’! XLIV.—Some Questions of Myriopod Nomenclature. : By R. I. Pocock. Pilsbry, Henry Augustus. 1901. "XLIII.—The Nomenclature of European Helices." The Annals and magazine of natural history; zoology, botany, and geology 8, 325–329. https://doi.org/10.1080/03745480109442929. 1. Leach’s Species of the Genus Geophilus. THE genus Geophilus, Leach, originally contained the fol- lowing species:—carpophagus, subterraneus, acuminatus, longicornis (Tr. Linn. Soe. xi. pp. 584-386, 1819). One of these must be the type of the genus. Longicornis was taken out by Newport (Proc. Zool. Soc. 1842, p. 180) as the type of Necrophleophagus. In 1845(Tr. Linn. Soe. xix. p. 429 &c.) Newport substituted <Arthronomalus for Necrophleophagus, and added the following species :—punctiventris, carpophagqus, similis (=carpophagus), Hopet, flavus, and opinatus. Since Newport expressly states that he changed Wecrophleophaqus for Arthronomalus, the two must be regarded as synonymous in the strictest possible sense of the term—that is to say, as having the same type species. It is superfluous, therefore, to pursue further the fate of the remaining species included under Arthronomalus with a view to ascertaining if one of This file was generated 1 April 2024 at 01:29 UTC Copyright & Reuse This document was created from content at the Biodiversity Heritage Library, the world's largest open access digital library for biodiversity literature and archives. Visit BHL at https://www.biodiversitylibrary.org. This file was generated 1 April 2024 at 01:29 UTC
https://openalex.org/W2953639234	https://www.e3s-conferences.org/articles/e3sconf/pdf/2019/18/e3sconf_isg2019_11019.pdf	English	null	Micro-continuum modelling of injection strategies for microbially induced carbonate precipitation	E3S web of conferences	2,019	cc-by	3,326	1 Introduction to MICP how improvement in soil strength is related to CaCO3 content (Fig. 1). However, only a limited number of field- scale trials have been carried out, each in quite different environments with different objectives and, consequently, differing treatment strategies [4,6–8]. Microbially Induced Carbonate Precipitation (MICP) is a naturally occurring process in which the by-products of bacteria growth and respiration create ideal chemical conditions for calcium carbonate to form as a mineral. We use the well-studied urea hydrolysis pathway (overall pathway shown in equations 1 & 2) to create calcium carbonate in-situ as an alternative grout with novel engineering properties. The inputs for this process are urea (a common fertiliser), a calcium source (e.g. calcium chloride), and ureolytically active bacteria, most commonly Sporosarcina pasteurii [1]. 𝑢𝑢𝑢𝑢𝑤𝑤𝑤𝑤𝑤𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 Fig. 1. Relationship between CaCO3 content and unconfined compressive strength, taken from the literature [9-20]. 𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢+ 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤+ 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 → 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐+ 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 (1) 𝐶𝐶𝐶𝐶(𝑁𝑁𝐻𝐻2)2 + 2𝐻𝐻2𝑂𝑂+ 𝐶𝐶𝑎𝑎2+ 𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢 → 𝐶𝐶𝐶𝐶𝐶𝐶𝑂𝑂3 + 2𝑁𝑁𝐻𝐻4 + (2) 𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢+ 𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤+ 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 → 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐+ 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 (1) 𝐶𝐶𝐶𝐶(𝑁𝑁𝐻𝐻2)2 + 2𝐻𝐻2𝑂𝑂+ 𝐶𝐶𝑎𝑎2+ 𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢 → 𝐶𝐶𝐶𝐶𝐶𝐶𝑂𝑂3 + 2𝑁𝑁𝐻𝐻4 + (2) (2) Fig. 1. Relationship between CaCO3 content and unconfined compressive strength, taken from the literature [9-20]. The advantages of using MICP over traditional cement and chemical grouts are 1) the input solutions have a near water-like viscosity meaning low injection pressure equipment is required and MICP can be used for near- surface ground improvement without risk of ground heave, 2) MICP could be more environmentally friendly with a potentially lower carbon footprint than cement, and less toxic than chemical grouts, and 3) it is possible to create impermeable barriers or to optionally increase strength without significantly reducing porosity and drainage. In order for MICP to progress from a lab-scale process to a commercially viable alternative ground treatment option, more field trials are required. To maximise the effectiveness of these future field trials we must first narrow down the extensive range of variables (relating to environmental conditions, what we inject, and how we inject it) into a handful of the most promising injection strategies. To do this, we have turned to numerical modelling of the MICP process with the aim of identifying strategies that make the most efficient use of reagents, make the most efficient use of time, and produce the most uniform and controllable treatment. Specifically, we model different urea/calcium concentrations, different reaction times, and three different types of ureolytic bacteria. James M. Minto1, Grainne El Mountassir1, and Rebecca J. Lunn1 1 University of Strathclyde, Department of Civil & Environmental Engineering, UK Abstract. Microbially induced carbonate precipitation is a promising technique for ground improvement. In order for MICP to progress from a lab-scale process to a commercially viable alternative ground improvement option, a combination of field-trials and field-scale modelling of the process is required. We present the results of a field-scale model in which differing injection strategies are evaluated and find that longer treatment times make more efficient use of reagents, but may come with higher operational costs, and that utilising multiple strains of bacteria with different reaction kinetics and transport properties may improve efficiency. © 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/). Corresponding author: james.minto@strath.ac.uk Corresponding author: james.minto@strath.ac.uk © 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/). E3S Web of Conferences 92, 11019 (2019) IS-Glasgow 2019 E3S Web of Conferences 92, 11019 (2019) IS-Glasgow 2019 https://doi.org/10.1051/e3sconf/20199211019 1 Introduction to MICP MICP appears to be a promising technique for reducing water flow in radioactive waste geological disposal facilities [2], for reducing leakage from oil, gas and carbon capture & storage wells [3,4], and as a ground improvement technique [5], particularly where near- surface treatment is required or where existing soil cannot be removed from site. MICP has been well studied in the lab. From these small-scale experiments we have learned, for example, E3S Web of Conferences 92, 11019 (2019) IS-Glasgow 2019 https://doi.org/10.1051/e3sconf/20199211019 Fig. 3. Field-scale model with location of wells and initial porosity distribution. 2 Model description The model has been implemented in the computational fluid dynamics software OpenFOAM. Fluid flow is based on the single-phase (fully saturated) Navier-Stokes equations with an additional term that accounts for resistance in porous media. In this way, the model can simulate fluid flow through fractures in rock, and in soil at both pore-scale and continuum-scale, hence the name micro-continuum modelling. Reactive transport of bacteria, urea, calcium, and the ammonium and carbonate by-products are included. Bacteria transport is a function of soil type, bacteria characteristics (size, stickiness), and velocity with less bacterial attachment in areas of high velocity. When urea (injected after the bacteria) reaches bacterial cells which have attached within the porous media, the urea is hydrolysed following the first-order Michaelis-Menten kinetics producing ammonium and carbonate. When calcium and carbonate mix, they precipitate as CaCO3 on the first surface encountered. Fig. 3. Field-scale model with location of wells and initial porosity distribution. Fig. 3. Field-scale model with location of wells and initial porosity distribution. Our field-scale test model consisted of a nine well array with 3m well spacing in which reagents are injected through the outer eight wells, and extracted from the central well. Initial conditions are a random porosity distribution (Fig. 3). the first surface encountered. Fig. 2. Scanning electron microscope image of microbially induced CaCO3. The rod shaped indentations are the remnants of bacterial cells that became encapsulated in the growing CaCO3 crystals. Fig. 4. Example of potential treatment strategies that can be modelled. Fig. 4. Example of potential treatment strategies that can be modelled. Fig. 2. Scanning electron microscope image of microbially induced CaCO3. The rod shaped indentations are the remnants of bacterial cells that became encapsulated in the growing CaCO3 crystals. Model boundary conditions are flexible enough to allow simultaneous injection and extraction from multiple wells (Fig. 4), as well as monitoring the cumulative concentrations of reagents entering and exiting the model through the wells and the four side boundaries. The total mass of CaCO3 precipitating within an area of interest extending 3m around each well is also monitored. The precipitated CaCO3 is non-transportable and occupies some of the pore space resulting in a lower permeability and a redirection of flow in subsequent injections of reagents. The CaCO3 also encapsulates attached bacteria, cutting them off from the urea and ending the reaction (Fig. 2). For this reason, multiple cycles of bacteria followed by urea/calcium cementing solution are injected until sufficient CaCO3 has been precipitated for effective ground improvement. Variables investigated were 1) the concentration of urea and calcium in the cementing solution (with a low concentration of 0.1 M and a high concentration of 0.4 M), 2) the length of time between cycles (between 2 and 22 hours) and 3) the type of ureaolytic bacteria. The model was calibrated with experimental results from batch experiments [21] in which urea hydrolysis over time was measured along with the final mass of CaCO3 produced. The standard bacteria was S. pasteurii with high ureaolytic activity (10mM urea/min/OD) and high Michaelis-Menten half saturation constant (301 mM). The second has the properties of a bacterium isolated from soil by Graddy et al. [22] designated LS57 with a lower activity of 5.6 mM urea/min/OD, but also a lower 2 2 E3S Web of Conferences 92, 11019 (2019) IS-Glasgow 2019 https://doi.org/10.1051/e3sconf/20199211019 saturation constant of 0.3 M for S. pasteurii (see solid lines in Fig. 6 where urea concentration was 0.1 M). In simulations where the urea concentration was 0.4 M (dashed lines in Fig. 6), replacing S. pasteurii with LS57 marginally reduced the amount of CaCO3 that precipitated over the first 12 hours of the cycle. half saturation constant of 64 mM. The lower half saturation constant of LS57 means that the reaction rate does not slow down as much at low urea concentrations (see Fig. 5). Run Urea/ Ca [M] Bacterial composition CaCO3 mass [kg] Urea utilization efficiency [%] 6 0.1/0.1 S.pasteurii 194 75.3% 7 0.2/0.2 S.pasteurii 333 64.6% 8 0.4/0.4 S.pasteurii 485 47.1% 9 0.1/0.1 S.pasteurii/LS57 206 80.1% 10 0.2/0.2 S.pasteurii/LS57 343 66.6% 11 0.4/0.4 S.pasteurii/LS57 489 47.5% 12 0.1/0.1 LS57 216 83.9% 13 0.2/0.2 LS57 351 68.2% 14 0.4/0.4 LS57 492 47.8% 15 0.1/0.1 S.pasteurii/ low attach LS57 240 93.0% 16 0.2/0.2 S.pasteurii/ low attach LS57 430 83.5% 17 0.4/0.4 S.pasteurii/ low attach LS57 634 61.6% Fig. 5. Urea hydrolysis kinetics for the two bacterial strains. The third bacterium was a hypothetical strain with urea hydrolysis properties similar to LS57, but with different physical cell properties (i.e. smaller cells that produce less extracellular polymeric substances/biofilm) hence can be transported further before attaching within the porous media. A special case is Run 17 where the urea concentration was 0.4 M, but LS57 was assumed to have different physical properties resulting in lower attachment. This case resulted in the greatest amount of CaCO3 and a significantly higher efficiency. The impact of altering bacterial attachment properties can be seen in Fig. 7. case resulted in the greatest amount of CaCO3 and a significantly higher efficiency. The impact of altering bacterial attachment properties can be seen in Fig. 7. Table 2. Field-scale simulation results for different bacterial strains. Mass of CaCO3 measured at the end of a single 24 hr injection cycle. Run Urea/ Ca [M] Bacterial composition CaCO3 mass [kg] Urea utilization efficiency [%] 6 0.1/0.1 S.pasteurii 194 75.3% 7 0.2/0.2 S.pasteurii 333 64.6% 8 0.4/0.4 S.pasteurii 485 47.1% 9 0.1/0.1 S.pasteurii/LS57 206 80.1% 10 0.2/0.2 S.pasteurii/LS57 343 66.6% 11 0.4/0.4 S.pasteurii/LS57 489 47.5% 12 0.1/0.1 LS57 216 83.9% 13 0.2/0.2 LS57 351 68.2% 14 0.4/0.4 LS57 492 47.8% 15 0.1/0.1 S.pasteurii/ low attach LS57 240 93.0% 16 0.2/0.2 S.pasteurii/ low attach LS57 430 83.5% 17 0.4/0.4 S.pasteurii/ low attach LS57 634 61.6% Fig. 6. Field-scale results showing total mass of CaCO3 precipitated during a single 24 hr injection cycle for all 4 bacteria strain combinations (see Table 2) at cementing solution concentrations of 0.1 M (solid lines) and 0.4 M (dashed lines). the porous media. Fig. 5. Urea hydrolysis kinetics for the two bacterial strains. Table 2. Field-scale simulation results for different bacterial strains. Mass of CaCO3 measured at the end of a single 24 hr injection cycle. Table 2. Field-scale simulation results for different bacterial strains. Mass of CaCO3 measured at the end of a single 24 hr injection cycle. 3 Model results All field-scale models were run with boundary conditions of fixed flow rate inlet, constant pressure outlets. The models indicated that increasing the reaction period between treatment cycles resulted in more CaCO3 precipitating and increased efficiency (where efficiency was measured as the amount of urea converted to CaCO3), but this came at the expense of longer treatment times (see Table 1). Decreasing the cementing solution concentration increased efficiency, but decreased the total mass of CaCO3. Table 1. Field-scale simulation results for reaction duration and cementing solution concentration for S.pasteurii. Mass of CaCO3 measured at the end of 10 injection cycles. CaCO3 measured at the end of 10 injection cycles. Run Reaction period [hrs] Urea/ Ca [M] Treatment time [days] CaCO3 mass [kg] Urea utilization efficiency [%] 1 4 0.4/0.4 2.65 2,887 28.0% 2 12 0.4/0.4 5.98 3,721 36.1% 3 2 0.4/0.4 1.81 2,460 23.9% 4 4 0.2/0.2 2.65 2,303 44.7% 5 12 0.2/0.2 5.98 2,762 53.7% Fig. 6. Field-scale results showing total mass of CaCO3 precipitated during a single 24 hr injection cycle for all 4 bacteria strain combinations (see Table 2) at cementing solution concentrations of 0.1 M (solid lines) and 0.4 M (dashed lines). The models also indicated that substituting some or all of the S. pasteurii with the LS57 bacterium resulted in more CaCO3 precipitating and a greater efficiency (Table 2), but only where the initial urea concentration was significantly lower than the Michaelis-Menten half 3 https://doi.org/10.1051/e3sconf/20199211019 E3S Web of Conferences 92, 11019 (2019) IS-Glasgow 2019 E3S Web of Conferences 92, 11019 (2019) Fig. 7. Field-scale results showing CaCO3 distribution after one injection cycle with a urea concentration of 0.4 M for a) S. pasteurii, and b) low attachment LS57. the injection wells where urea concentrations are highest, whilst LS57 attaches further away where it can make efficient use of the leftover urea that makes it to these outer edges. Modelling shows that an approach of combining different bacteria strains with different attachment properties may make more efficient use of the urea. It has also been suggested that co-cultures of bacteria can increase CaCO3 precipitation by providing more nucleation sites [23,24] or prove to be more robust across a range of environmental conditions [25]. However, these benefits would have to be weighed against the extra costs required to optimally grow, store, and transport multiple types of bacteria compared with S. pasteurii alone. Fig. 7. 3 Model results Field-scale results showing CaCO3 distribution after one injection cycle with a urea concentration of 0.4 M for a) S. pasteurii, and b) low attachment LS57. References In the field-scale model we see that longer reaction periods made more efficient use of urea. However, this came with diminishing returns for two reasons: 1) most of the bacteria become encapsulated in the early part of each injection cycle hence the reaction rate drops considerably within a few hours, and 2) the Michaelis-Menten reaction kinetics mean that as urea concentration decreases, the rate of urea hydrolysis further decreases. 1. J.H. Yoon, K.C. Lee, N. Weiss, Y.H. Kho, K.H. Kang, Y.H. Park, Sporosarcina aquimarina sp. nov., a bacterium isolated from seawater in Korea, and transfer of Bacillus globisporus (Larkin and Stokes 1967), Bacillus psychrophilus (Nakamura 1984) and Bacillus pasteurii (Chester 1898) to the genus Sporosarcina as Sporosa, Int. J. Syst. Evol. Microbiol. 51 (2001) 1079–86. http://www.ncbi.nlm.nih.gov/pubmed/11411676. The most efficient strategy based on utilization of urea and bacteria would be long cementation periods (in excess of 12 hours) and low urea/ calcium concentrations (lower than 0.2 M) as, even under these conditions, the supply of urea and un-encapsulated bacteria were not exhausted before the next injection cycle. However, although less efficient in terms of reagents, a two-hour cementation period with a concentration of 0.4 M produced a comparable amount of CaCO3 in approximately one third the time. 2. J.M. Minto, E. MacLachlan, G. El Mountassir, R.J. Lunn, Rock fracture grouting with microbially induced carbonate precipitation, Water Resour. Res. 52 (2016) 8827–8844. doi:10.1002/2016WR018884. 3. A.B. Cunningham, A.J. Phillips, E. Troyer, E. Lauchnor, R. Hiebert, R. Gerlach, L. Spangler, Wellbore leakage mitigation using engineered biomineralization, Energy Procedia. 63 (2014) 4612–4619. doi:10.1016/j.egypro.2014.11.494. As much of the costs associated with ground improvement are due to staff time and equipment that is tied-up on site, together with heavy penalties for delays in the construction schedule, we suspect that there are situations where an engineering contractor would find it economical to optimise MICP ground improvement to minimise treatment time, rather than maximise reagents efficiency. 4. A.J. Phillips, A.B. Cunningham, R. Gerlach, R. Hiebert, C. Hwang, B.P. Lomans, J. Westrich, C. Mantilla, J. Kirksey, R.A. Esposito, L. Spangler, Fracture Sealing with Microbially-Induced Calcium Carbonate Precipitation: A Field Study, Environ. Sci. Technol. (2016) acs.est.5b05559. doi:10.1021/acs.est.5b05559. 4.2 Bacterial strain combinations S. pasteurii is the most commonly utilised bacteria for MICP due to its comparably high ureolytic activity. We find that when urea concentrations are high, S. pasteurii has the desirable trait of producing more CaCO3 in a shorter period of time than strains with a lower activity. 5. J.T. DeJong, K. Soga, S. a Banwart, W.R. Whalley, T.R. Ginn, D.C. Nelson, B.M. Mortensen, B.C. Martinez, T. Barkouki, Soil engineering in vivo: harnessing natural biogeochemical systems for sustainable, multi-functional engineering solutions, J. R. Soc. Interface. 8 (2010) 1–15. doi:10.1098/rsif.2010.0270. However, if maximum reagent efficiency is required, at some point urea concentration must drop below S. pasteurii’s optimum concentration. At this point, other less active bacteria may precipitate more CaCO3 in a given period of time if, like strain LS57, they have a lower Michaelis-Menten half saturation constant that allows them to be more efficient at lower urea concentrations. 6. L.A. van Paassen, M.P. Harkes, G.A. Van Zwieten, W.H. van der Zon, W.R.L. Van Der Star, M.C.M. van Loosdrecht, Scale up of BioGrout: A biological ground reinforcement method, Proc. 17th Int. Conf. Soil Mech. Geotech. Eng. Acad. Pract. Geotech. Eng. 3 (2009) 2328–2333. doi:10.3233/978-1- 60750-031-5-2328. Finally, combining S. pasteurii with a hypothetical bacterium with the reaction kinetics of LS57, but with different attachment properties, produces the most CaCO3. This is because the two strains are spatially distributed in different areas: S. pasteurii attaches close to 4 E3S Web of Conferences 92, 11019 (2019) IS-Glasgow 2019 https://doi.org/10.1051/e3sconf/20199211019 18. D. Terzis, L. Laloui, 3-D micro-architecture and mechanical response of soil cemented via microbial-induced calcite precipitation, Sci. Rep. 8 (2018) 1416. doi:10.1038/s41598-018-19895-w. 7. M.G. Gomez, B.C. Martinez, J.T. DeJong, C.E. Hunt, L.A. DeVlaming, D.W. Major, S.M. Dworatzek, Field-scale bio-cementation tests to improve sands, Proc. Inst. Civ. Eng. - Gr. 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https://openalex.org/W4392450333	http://altifani.org/index.php/altifani/article/download/518/258	Indonesian	null	Pelatihan Pembuatan Salep Untuk Infeksi Kulit dari Ekstrak Daun Karamunting (Rhodomyrtus Tomentosa) Di Desa Tanjung Baru, Ogan Ilir	Jurnal Altifani Penelitian dan Pengabdian kepada Masyarakat	2,024	cc-by-sa	3,200	Pelatihan pembuatan salep untuk infeksi kulit dari ekstrak daun karamunting (Rhodomyrtus tomentosa) di desa Tanjung Baru, Ogan Ilir Ferlinahayati1, Addy Rachmat1, Hermansyah1, Elfita1, Poedji Loekitowati Hariani1 3Program Studi Kimia, Fakultas Matematika dan Ilmu Pengetahuan Alam, Universitas Sriwijaya e-mail: puji_lukitowati@mipa.unsri.ac.id, ferlinahayati@mipa.unsri.ac.id, addy_rachmat@unsri.ac.id, hermansyah@unsri.ac.id, elfita_kimia@unsri.ac.id Revised: 26.12.2023 Accepted: 05.01.2024 Available online: 25.01.2024 Accepted: 05.01.2024 Revised: 26.12.2023 Abstract: The skin serves as the primary defense mechanism protecting the body from diseases. Skin infections can be caused by microorganisms, such as bacteria, viruses, fungi, or parasites. The karamunting plant (Rhodomyrtus tomentosa) has traditionally been utilized by the community for healing purposes, including the treatment of wounds. This community service initiative aims to provide training in the production of ointments for skin infection treatment using vaseline-based ethanol extracts from karamunting leaves. The community service activities were conducted in Tanjung Baru village, Indralaya Utara sub-district, Ogan Ilir district. The target audience consisted of 30 PKK mothers and young women. The stages of the activity included: (i) education on skin infections, their causes, transmission, and treatment; (ii) training on creating ethanol extracts from karamunting leaves and producing vaseline-based ointments; and (iii) evaluating the outcomes of the activity. The results of the activity evaluation, obtained through some questions, revealed that 97.62 % of participants provided correct and highly accurate answers regarding the material covered. The test results assessing the liking and comfort levels of the ointment demonstrated that 82.64 % of participants expressed a strong preference for the aroma, appearance, color, comfort, and stickiness when using the ointment. This suggests that the public readily accepts this ointment for skin infections as an alternative treatment. Keywords: karamunting leaves, ethanol extract, ointments, skin infection Abstrak: Kulit memiliki fungsi sebagai pertahanan pertama untuk melindungi tubuh dari penyakit. Infeksi kulit dapat disebabkan oleh mikroorganisme, seperti bakteri, virus, jamur, atau parasit. Tanaman karamunting (Rhodomyrtus tomentosa) secara tradisional telah digunakan masyarakat untuk pengobatan termasuk pengobatan luka. Kegiatan pengabdian masyarakat ini bertujuan memberikan pelatihan pembuatan salep untuk pengobatan infeksi kulit dari ekstrak etanol daun karamunting berbasis vaselin. Kegiatan pengabdian masyarakat dilakukan di desa Tanjung Baru, kecamatan Indralaya Utara, kabupaten Ogan Ilir. Khalayak sasaran adalah ibu-ibu PKK dan remaja putri sebanyak 30 orang. Tahapan kegiatan yaitu (i) penyuluhan tentang penyakit infeksi kulit, penyebab infeksi kulit, penularan dan pengobatan, (ii) pelatihan pembuatan ekstrak etanol dari daun karamunting, pembuatan salep berbasis vaselin dan (iii) evaluasi hasil kegiatan. Hasil evaluasi kegiatan dengan memberikan berbagai pertanyaan setelah kegiatan menunjukkan 97,62 % peserta menjawab tepat dan sangat tepat materi yang diberikan. Pelatihan pembuatan salep untuk infeksi kulit dari ekstrak daun karamunting (Rhodomyrtus tomentosa) di desa Tanjung Baru, Ogan Ilir Hasil uji tingkat kesukaan dan kenyamanan penggunaan salep menunjukkan sebanyak 82,64 % peserta menyatakan sangat suka terhadap aroma, bentuk, warna, kenyamanan dan daya lekat ketika salep digunakan. Hal ini mengindikasikan bahwa salep untuk infeksi kulit ini diterima dengan baik oleh masyarakat sebagai alternatif pengobatan penyakit infeksi kulit. Kata kunci: daun karamunting, ekstrak etanol, salep, infeksi kulit JURNAL ALTIFANI JURNAL ALTIFANI Vol. 4, No. 1 - Januari 2024, Hal. 29-35 DOI: 10.59395/altifani.v4i1.518 Penelitian dan Pengabdian kepada Masyarakat Penelitian dan Pengabdian kepada Masyarakat Penelitian dan Pengabdian kepada Masyarakat Pelatihan pembuatan salep untuk infeksi kulit dari ekstrak daun karamunting (Rhodomyrtus tomentosa) di desa Tanjung Baru, Ogan Ilir Penelitian dan Pengabdian kepada Masyarakat terutama pada anak-anak. Padahal bagian kulit yang telah terinfeksi bakteri secara berkelanjutan tanpa pengobatan jika sembuh akan menimbulkan bekas yang kurang baik. Penggunaan obat tradisional sebagai sediaan bioaktif memiliki kelebihan yaitu mudah diperoleh, mudah membuatnya, dapat disimpan lama dan tidak menimbulkan resistensi. Tanaman karamunting (Rhodomyrtus tomentosa) termasuk famili Myrtaceae. Kandungan fitokimia pada tanaman karamunting sebagai sumber potensial agen antimikroba dan imunomodulator (Hmoteh et al., 2018). Secara tradisional tanaman ini telah digunakan di negara- negara Asia Tenggara termasuk Indonesia untuk meredakan berbagai gejala peradangan seperti diare, ginekopati, infeksi saluran kemih, dan infeksi luka (Siriyong et al., 2020). Tumbuhan karamunting merupakan tumbuhan perdu yang hidup di semak-semak belukar, tinggi kira-kira 1-2 meter, bunga berwarna merah muda. Tanaman ini mudah tumbuh dan berkembang biak. Seluruh bagian tanaman ini (daun, akar, tunas dan buah) telah digunakan dalam pengobatan tradisional dalam pengobatan sejak lama. Kerusakan jaringan dan luka inflamasi disebabkan oleh infeksi bakteri dapat disembuhkan menggunakan ekstrak daun karamunting. Ekstrak etanol daun karamunting memiliki nilai konsentrasi hambat mnimum (KHM) sebesar 16–64 µg/mL terhadap bakteri Staphylococcal (Mordmuang et al., 2015). Hasil penelitian lain menunjukkan bahwa ekstrak daun karamunting dapat dibuat sediaan fitofarmaka yaitu 10 % ekstrak ekstrak etanol dalam basis vaselin dapat menyembuhkan infeksi kulit pada kelinci selama 5 hari pengobatan (Dachriyanus et al., 2002). Kandungan kimia tanaman karamunting antara lain triterpenoid, flavonoid, fenol, meroterpenoid dan unsur mikro (Ferlinahayati et al., 2020; Zhao et al., 2020). Diantara bahan-bahan tersebut, Rhodomirton merupakan senyawa bioaktif yang paling potensial dengan berbagai aktivitas farmakologi (Lai et al., 2013; Zhao et al., 2020). Beberapa penelitian menunjukkan bahwa Rhodomirton merupakan agen antibakteri untuk pengobatan infeksi bakteri Gram-positif (Mitsuwan et al., 2017; Saeloh et al., 2017). Hasil penelitian Sianglum et al (2018) menunjukkan bahwa rhodomyrtone dapat meningkatkan permeabilisasi membran methicillin-resistant Staphylococcus aureus (MRSA), perubahan dinding sel, dan integritas membran sel. Sebagai dampaknya terjadi kebocoran komponen sitoplasma, lisis sel, dan sel bakteri mengalami kerusakan. Penelitian lain menunjukkan bahwa Rhodomyrtone, memiliki anti- staphylococcal biofilm (Saising et al., 2018), dan aktivitas antijamur terhadap Candida albicans (Limsuwan et al., 2011), antioksidan (Lavanya et al., 2012), dan aktivitas imunomodulator (Hmoteh et al., 2016). Kegiatan pengabdian pada masyarakat ini memberikan pendampingan dan pelatihan pembuatan serta penggunaan obat tradisional dari ekstrak daun karamunting sebagai sediaan bioaktif yang dibuat menjadi salep. Kelebihan dari sediaan ini adalah bahan baku mudah diperoleh, mudah membuatnya, dapat disimpan lama dan tidak menimbulkan resistensi. Penelitian dan Pengabdian kepada Masyarakat Sebagai khalayak sasaran pelatihan adalah karang taruna dan ibu ibu PKK di desa Tanjung Baru. JURNAL ALTIFANI Vol. 4, No. 1 - Januari 2024, Hal. 29-35 DOI: 10.59395/altifani.v4i1.518 1. PENDAHULUAN Infeksi kulit adalah penyakit yang disebabkan oleh mikroorganisme, seperti bakteri, virus, jamur, atau parasit. Infeksi kulit dapat menimbulkan beragam gejala, mulai dari gatal-gatal sampai luka yang disertai nyeri. Salah satunya fungsi kulit adalah melindungi bagian dalam tubuh dari dari lingkungan sekitar seperti agen infeksius, radiasi sinar ultraviolet, cedera, dan infeksi (Hu et al., 2018). Penyakit yang menyerang kulit bisa menimbulkan gejala yang amat mengganggu dan mungkin baru terasa saat keluhannya sudah telanjur parah. Penyakit infeksi kulit umumnya disebabkan oleh mikroorganisme. Staphylococcus aureus (S. aureus) adalah salah satu bakteri paling umum hidup pada luka kronis, berpotensi menimbulkan risiko infeksi luka, terutama jika menghasilkan faktor virulensi yang melebihi faktor inangnya (Bessa et al., 2015). Penyakit infeksi kulit dapat disebabkan karena infeksi primer oleh satu jenis bakteri atau infeksi sekunder, yang disebabkan beberapa macam bakteri yang terdapat pada jaringan kulit yang rusak (Atta et al., 2023). Desa Tanjung Baru terletak di kecamatan Inderalaya Utara, kabupaten Ogan Ilir. Wilayah ini sebagian dikelilingi rawa-rawa. Penyakit infeksi kulit masih banyak ditemukan di daerah tersebut E-ISSN: 2774-6240 29 Penelitian dan Pengabdian kepada Masyarakat Gambar 1. Metode pelaksanaan kegiatan pengabdian Masyarakat di desa Tanjung Baru JURNAL ALTIFANI JURNAL ALTIFANI Penelitian dan Pengabdian kepada Masyarakat Vol. 4, No. 1 - Januari 2024, Hal. 29-35 DOI: 10.59395/altifani.v4i1.518 Gambar 1. Metode pelaksanaan kegiatan pengabdian Masyarakat di desa Tanjung Baru Vol. 4, No. 1 - Januari 2024, Hal. 29-35 DOI: 10.59395/altifani.v4i1.518 Penelitian dan Pengabdian kepada Masyarakat 2. METODE Kegiatan pengabdian yang dilakukan meliputi beberapa tahap yaitu (i) penyuluhan tentang penyakit infeksi kulit, penyebab, penularan dan pengobatan, (ii) pelatihan ekstraksi bahan bioaktif dari daun karamunting dan pembuatan salep obat infeksi kulit, dan (iii) pelatihan cara penggunaan, dosis dan penyimpanan salep, dan (iv) evaluasi kegiatan dengan memberikan kuisoner. Kegiatan diikuti oleh ibu-ibu PKK dan remaja putri sebanyak 30 orang. Gambar 1 menunjukkan metode pelaksanaan kegiatan pengabdian Masyarakat. E-ISSN: 2774-6240 30 3. HASIL DAN PEMBAHASAN Kegiatan pengabdian masyarakat dilaksanakan di desa Tanjung Baru, kecamatan Indralaya Utara, kabupatan Ogan Ilir. Peserta kegiatan adalah ibu-ibu PKK dan remaja putri yang berjumlah 30 orang. Kegiatan yang pertama adalah penyuluhan tentang penyakit kulit, penyebabnya, penularan dan pengobatan. Beberapa kebiasaan atau faktor lingkungan tertentu dapat berkontribusi pada risiko terjadinya penyakit kulit, seperti kurang menjaga kebersihan badan, kebiasaan menggaruk kulit badan, paparan sinar matahari yang berlebihan, kebiasaan saling bertukar pakaian, handuk dll. Kekebalan tubuh yang rendah juga menjadi pemicu terjadinya infeksi kulit, karena sistem kekebalan tubuh yang melemah atau terganggu dapat membuat seseorang lebih rentan. Infeksi ini ditandai dengan kulit kering dan bersisik, bercak merah, kulit terluka (borok), ruam yang disertai rasa gatal atau nyeri dll. Gambar 2 menunjukkan kegiatan penyuluhan yang dilakukan oleh tim pengabdian masyarakat. Masyarakat menyimak dan tertarik dengan materi yang diberikan. Gambar 2. Kegiatan penyuluhan tentang penyakit infeksi kulit Kegiatan pelatihan dilakukan dengan membagi peserta menjadi 3 kelompok, masing-masing kelompok didampingi tim pengabdian dan mahasiswa. Pelatihan pembuatan ekstrak daun karamunting menggunakan pelarut etanol dengan cara maserasi. Perbandingan simplisia (daun karamunting kering) dengan etanol adalah 1:3. Ekstraksi dilakukan selama 3 kali, tiap tahap ekstraksi dilakukan selama 2 hari. Selanjutnya ampas dikeluarkan dan ekstrak etanol cair diuapkan sampai didapatkan ekstrak kental, menggunakan penangas dengan api kecil. Gambar 3 menunjukkan simplisia dan ekstrak etanol daun karamunting. Gambar 2. Kegiatan penyuluhan tentang penyakit infeksi kulit Gambar 2. Kegiatan penyuluhan tentang penyakit infeksi kulit Kegiatan pelatihan dilakukan dengan membagi peserta menjadi 3 kelompok, masing-masing kelompok didampingi tim pengabdian dan mahasiswa. Pelatihan pembuatan ekstrak daun karamunting menggunakan pelarut etanol dengan cara maserasi. Perbandingan simplisia (daun karamunting kering) dengan etanol adalah 1:3. Ekstraksi dilakukan selama 3 kali, tiap tahap ekstraksi dilakukan selama 2 hari. Selanjutnya ampas dikeluarkan dan ekstrak etanol cair diuapkan sampai didapatkan ekstrak kental, menggunakan penangas dengan api kecil. Gambar 3 menunjukkan simplisia dan ekstrak etanol daun karamunting. E-ISSN: 2774-6240 31 JURNAL ALTIFANI Vol. 4, No. 1 - Januari 2024, Hal. 29-35 DOI: 10.59395/altifani.v4i1.518 Penelitian dan Pengabdian kepada Masyarakat Penelitian dan Pengabdian kepada Masyarakat Gambar 3. (a) Simplisia dan (b) Ekstrak etanol daun karamunting (a) (b) (a) (b) Gambar 3. (a) Simplisia dan (b) Ekstrak etanol daun karamunting Kegiatan selanjutnya adalah pelatihan pembuatan salep untuk infeksi kulit. Bahan yang diperlukan adalah ekstrak etanol daun karamunting dan vaselin. Perbandingan antara ekstrak etanol daun karamunting dan vaselin adalah adalah 1:10. Salep yang sudah jadi tersebut disimpan dalam botol yang telah steril. Cara mensterilkan dengan cara memanaskan botol dalam air panas selama pada suhu ± 60o C selama 15 menit. Salep dari ekstrak etanol daun karamunting ini, jika proses penyimpanan tepat maka dapat digunakan sampai 3 bulan. Gambar 4 menunjukkan kegiatan pelatihan pembuatan salep infeksi kulit. Gambar 4. Kegiatan pelatihan dan pendampingan pembuatan salep obat infeksi kulit Gambar 4. Kegiatan pelatihan dan pendampingan pembuatan salep obat infeksi kulit S. aureus merupakan bakteri gram positif yang dapat menyebabkan infeksi pada jaringan lunak, kulit, sendi, tulang, dan abses. Bakteri ini seringkali ditemukan pada kulit dan selaput lendir manusia dan dapat menyebabkan berbagai infeksi kulit, termasuk impetigo, selulitis, dan folikulitis. S. aureus juga dapat menjadi penyebab infeksi kulit yang lebih serius jika bakteri tersebut memasuki aliran darah (Hannan et al., 2008). Pengobatan infeksi kulit harus dilakukan dengan tepat agar kulit yang terinfeksi dapat sembuh. Tim pengabdian memberikan penjelasan cara penyimpanan, cara E-ISSN: 2774-6240 32 JURNAL ALTIFANI JURNAL ALTIFANI Vol. 4, No. 1 - Januari 2024, Hal. 29-35 DOI: 10.59395/altifani.v4i1.518 DOI: 10.59395/altifani.v4i1.518 Penelitian dan Pengabdian kepada Masyarakat Tingkat kesukaan atau kenyaman penggunaan salep disajikan pada gambar 6. Jawaban terbanyak adalah sangat suka yaitu sebesar 82,64 % baik terhadap aroma, bentuk, warna, kenyamanan dan daya lekat salep. Masyarakat tidak ada yang menjawab netral, kurang suka bahkan tidak suka. Hal ini menunjukkan bahwa salep infeksi kulit dari ekstrak daun karamunting diterima baik oleh masyarakat. Gambar 6. Tingkat kesukaan peserta terhadap salep obat infeksi kulit Gambar 6. Tingkat kesukaan peserta terhadap salep obat infeksi kulit 4. KESIMPULAN S U Kegiatan pengabdian masyarakat yaitu pelatihan pembuatan salep untuk infeksi kulit yang dilakukan di desa Tanjung Baru, kecamatan Indralaya Utara, Kabupaten Ogan Ilir telah berhasil: 1. Meningkatkan pengetahuan masyarakat tentang penyakit infeksi pada kulit, penye penularan dan pengobatan Meningkatkan pengetahuan masyarakat tentang penyakit infeksi pada kulit, penyebabny penularan dan pengobatan 2. Meningkatkan ketrampilan masyarakat membuat salep dari ekstrak etanol daun karamunting berbasis vaselin dan mampu menggunakannya untuk pengobatan infeksi kulit 3. Sebanyak 82,64 % masyarakat menyatakan sangat suka terhadap aroma, bentuk, warna, kenyamanan dan daya lekat produk salep untuk infeksi kulit Masyarakat menyatakan terimakasih atas pengetahuan dan ketrampilan yang telah diberikan oleh tim pengabdian pada masyarakat, untuk selanjutnya perlu pembinaan dari tenaga Kesehatan untuk selalu memotivasi masyarakat agar menjaga kesehatan kulit khususnya pada anak-anak. UCAPAN TERIMA KASIH Kegiatan pengabdian masyarakat ini dibiayai oleh Anggaran DIPA Badan Layanan Umum Universitas Sriwijaya Tahun Anggaran 2023. SP DIPA-023.17.2.67751512023, tanggal 10 Mei 2023. Sesuai dengan SK Rektor Nomor: 0007/UN9/SK.LP2M.PMl2023 tanggal 20 Juni 2023. Kegiatan pengabdian masyarakat ini dibiayai oleh Anggaran DIPA Badan Layanan Umum Universitas Sriwijaya Tahun Anggaran 2023. SP DIPA-023.17.2.67751512023, tanggal 10 Mei 2023. Sesuai dengan SK Rektor Nomor: 0007/UN9/SK.LP2M.PMl2023 tanggal 20 Juni 2023. JURNAL ALTIFANI Vol. 4, No. 1 - Januari 2024, Hal. 29-35 DOI: 10.59395/altifani.v4i1.518 Dachriyanus, Salni, Sargent, M. V., Skelton, B. W., Soediro, I., Sutisna, M., White, A., & Yulinah, E. (2002). Rhodomyrtone, an atibiotic from rhomyrtus tomentosa. Australian Journal of Chemistry, 55(3), 229-232. Penelitian dan Pengabdian kepada Masyarakat pengunaan dan dosis yang digunakan untuk pengobatan. Gambar 5 menyajikan cara penggunaan salep tersebut dan produk salep yang telah dikemas. Gambar 5. Cara penggunaan salep untuk infeksi kulit dan produk salep yang telah dikemas Gambar 5. Cara penggunaan salep untuk infeksi kulit dan produk salep yang telah dikemas Tahap akhir kegiatan adalah evaluasi terhadap kegiatan. Evaluasi meliputi (i) pemahaman terhadap penyakit infeksi kulit dan (ii) evaluasi tingkat pemahaman pembuatan salep dan (iii) tingkat kesukaan dan kenyamanan terhadap produk salep. Tabel 1 menunjukkan hasil evaluasi kepada 30 peserta setelah kegiatan pengabdian masyarakat berlangsung. Berdasarkan data tabel 1 tersebut, secara umum tampak bahwa peserta kegiatan dapat menjawab dengan tepat dan sangat tepat berbagai pertanyaan yang diberikan. Rata-rata jawaban tepat dan sangat tepat 97,62 %, sedangkan kurang tepat sebesar 2,38 %. Tabel 1. Evaluasi sebagai indikator keberhasilan kegiatan Hasil evaluasi Kurang tepat (%) Tepat (%) Sangat tepat (%) Masyarakat memahami pentingnya menjaga kesehatan kulit dan dampaknya jika terkena infeksi kulit - 86,67 13,33 Masyarakat memahami pentingnya mengobati penyakit infeksi kulit 6,67 76,67 16,67 Masyarakat memahami penggunaan tanaman untuk pengobatan penyakit infeksi kulit 3,33 80,00 16,67 Masyarakat dapat menjelaskan kegunaan ekstrak daun karamunting pada pembuatan salep 3,33 76,67 20,00 Masyarakat dapat menyebutkan cara ekstraksi daun karamunting secara maserasi dengan tepat - 73,33 26,67 Masyarakat dapat menyebutkan bahan-bahan yang digunakan dalam pembuatan salep - 13,33 86,67 Masyarakat dapat memahami cara mengemas, menyimpan dan menggunakan salep untuk infeksi kulit 3,33 50,00 46,67 Rata-rata 2,38 65,24 32,38 33 E-ISSN: 2774-6240 Bessa, L.J., Fazii, P., Di Giulio, M., & Cellini, L. (2015). 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Bacterial isolates from infected wounds and their antibiotic susceptibility pattern: some remarks about wound infection. International Wound Journal, 12, 47-52. Dachriyanus, Salni, Sargent, M. V., Skelton, B. W., Soediro, I., Sutisna, M., White, A., & Yulinah, E. (2002). Rhodomyrtone, an atibiotic from rhomyrtus tomentosa. Australian Journal of Chemistry, 55(3), 229-232. 34 E-ISSN: 2774-6240 JURNAL ALTIFANI Vol. 4, No. 1 - Januari 2024, Hal. 29-35 DOI: 10.59395/altifani.v4i1.518 Penelitian dan Pengabdian kepada Masyarakat W., Tipmanee, V., & Voravuthikunchai, S. P. (2017). Effects of rhodomyrtone on Gram-positive bacterial tubulin homologue FtsZ. Peer J, 5, 1-24. Saising, J., Nguyen, M. T, Härtner, T., Ebner, P., Bhuyan, A. A. M., Berscheid. A., Muehlenkamp, M., Schäkermann, S., Kumari, N., Maier, M.E., Voravuthikunchai, S. P., Bandow, J., Lang, F., Brötz-Oesterhelt, H., & Götz, F. (2018). Rhodomyrtone (Rom) is a membrane-active compound. 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https://openalex.org/W4381273418	https://zenodo.org/record/8058472/files/8.pdf	English	null	ISLAM AND GREEN TECHNOLOGY	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	3,542	ISLAM AND GREEN TECHNOLOGY Mohamad Zaidin Mohamad1, Noorsafuan Che Noh1, Mohd Faiz Hakimi Mat Idris2, A Sofyuddin Yusof2 and Berhanundin Abdullah2 Mohamad Zaidin Mohamad1, Noorsafuan Che Noh1, Mohd Faiz Hakimi Mat Idris2, Ahmad Fauzi Hasan2, Sofyuddin Yusof2 and Berhanundin Abdullah2 , N C N , , , Sofyuddin Yusof2 and Berhanundin Abdullah2 1. Research Institute for Islamic Products & Civilization (INSPIRE), Universiti Sultan Zainal Abidin, Gong Badak Campus, 21300 Kuala Nerus, Terengganu. 1. Research Institute for Islamic Products & Civilization (INSPIRE), Universiti Sultan Zainal A Campus, 21300 Kuala Nerus, Terengganu. 2. Faculty of Islamic Contemporary Studies, Universiti Sultan Zainal Abidin, Gong Badak Campus, 21300 Kuala Nerus, Terengganu. …………………………………………………………………………………………………….... Manuscript Info Abstract ……………………. ……………………………………………………………… Manuscript History Received: 23 March 2023 Final Accepted: 27 April 2023 Published: May 2023 Key words:- Green Technologhy, Islam, Ethic, Da‟wah, Principles Islam is a religion that emphasizes the importance of protecting the environment and preserving the earth‟s natural resources. With the rise of global warming and climate change, the need for green technology has become more pressing than ever before. This study aims to identify the Islamic perspective and implementation of such programs in Islamic history, especially during the era of Andalusia. This study is a qualitative approach with a research design of document analysis. The study‟s findings show some of the green technologies that are compatible with Islamic principles, such as renewable energy sources and sustainable agriculture. Finally, the article concludes by highlighting the need for a holistic approach to environmental protection that integrates both Islamic teachings and modern green technologies. Copy Right, IJAR, 2023,. All rights reserved. …………………………………………………………………………………………………….... Introduction:- Green technology is a technology that is developed to meet human needs and can operate more efficiently while ……………………………………………………………… Islam is a religion that emphasizes the importance of protecting the environment and preserving the earth‟s natural resources. With the rise of global warming and climate change, the need for green technology has become more pressing than ever before. This study aims to identify the Islamic perspective and implementation of such programs in Islamic history, especially during the era of Andalusia. This study is a qualitative approach with a research design of document analysis. The study‟s findings show some of the green technologies that are compatible with Islamic principles, such as renewable energy sources and sustainable agriculture. ISLAM AND GREEN TECHNOLOGY Finally, the article concludes by highlighting the need for a holistic approach to environmental protection that integrates both Islamic teachings and modern green technologies. Copy Right, IJAR, 2023,. All rights reserved. ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 11(05), 1338-1342 Journal Homepage: - www.journalijar.com Article DOI: 10.21474/IJAR01/17001 DOI URL: http://dx.doi.org/10.21474/IJAR01/17001 Int. J. Adv. Res. 11(05), 1338-1342 ISSN: 2320-5407 Int. J. Adv. Res. 11(05), 1338-1342 “Indeed, Allah „AzzaWa Jal loves it when one of you performs an action, that it is done diligently.” (al-Tabarani, 1995) The use of green technology can also be a form of perpetual charity, which is a good deed that continues to benefit others even after the doer has passed away. In Islam, perpetual charity is highly recommended and regarded as an investment for the afterlife (Harfiyah Abdel Haleem. 1999; Norizan et al., 2016). In summary, Islam strongly encourages the use of green technology as an effort to protect and preserve the environment, which is a gift from Allah SWT. Moreover, Islam also pays attention to the ethics of using green technology. Green technology must be used wisely and not harm the environment or human health. This is in line with Islamic principles that teach simplicity, orderliness, and justice in the use of natural resources. In Islam, green technology can be used to support da‟wah activities and the welfare of humanity. For example, the use of green technology in waste processing can help maintain environmental cleanliness, thus becoming a form of rewarding charity. g y However, Islam also emphasizes that green technology should not be made the primary goal, but only a means to achieve a greater goal, which is to preserve the environment and the welfare of humanity. g y However, Islam also emphasizes that green technology should not be made the primary goal, but only a means to achieve a greater goal, which is to preserve the environment and the welfare of humanity. To promote green technology, Islam also encourages the development of innovation and research in the field of green technology. In this regard, Islam teaches that science and technology must be applied wisely and responsibly, to provide greater benefits to human life and the environment (Sayem, 2021; Aburounia, Sexton, 2006; Ashtankar, 2016). Principles of Islam in Green Technology:- The principles of Islam for green technology can be explained as follows: The principles of Islam for green technology c 1. Justice in the use of natural resources. Islam teaches that humans should treat natural resources fairly and wisely. Natural resources provided by Allah SWT to humans must be used responsibly and not harm the environment. This is in line with the concept of sustainable development that prioritizes social, economic, and environmental justice (Muhammad A. S. &RijaSudirja, 2007). 1. Justice in the use of natural resources. Islam teaches that humans should treat natural resources fairly and wisely. Natural resources provided by Allah SWT to humans must be used responsibly and not harm the environment. This is in line with the concept of sustainable development that prioritizes social, economic, and environmental justice (Muhammad A. S. &RijaSudirja, 2007). 2. Simplicity and orderliness. Islam teaches that humans should live simply and not excessively use natural resources. This can be done by reducing the use of energy and chemicals that are harmful to the environment and human health. In addition, Islam also emphasizes the importance of orderliness in the management of natural resources to maintain ecosystem balance (Muhammad S.A. et al, 2021). 3. Obligation to protect the environment. Islam teaches that humans, as stewards on earth, have a duty to protect and preserve the environment. This is in line with the principles of Islamic environmental ethics that respect the existence of other living beings and encourage humans to maintain the cleanliness and beauty of nature (Wardah, 2022). 4. Development of innovation and research. Islam encourages the development of innovation and research in the field of green technology to produce more effective and efficient solutions in protecting the environment. This is in line with the principles of science and technology in Islam, which prioritize the wise and responsible application of science and technology (Mehran, 2011). 4. Development of innovation and research. Islam encourages the development of innovation and research in the field of green technology to produce more effective and efficient solutions in protecting the environment. This is in line with the principles of science and technology in Islam, which prioritize the wise and responsible application of science and technology (Mehran, 2011). Rasulullah SAW and Green Technology:- Introduction:- Green technology is a technology that is developed to meet human needs and can operate more efficiently while having a smaller environmental impact. In Islam, the natural environment is a gift from Allah SWT that must be protected and preserved to maintain the sustainability of human life and other creatures. Therefore, the Islamic view of green technology is very positive because it can help to protect the environment. The Islamic view of green technology can be found in various verses of the Quran and hadith, such as in Surah al- Baqarah verse 60 and Surah al-Rum verse 41. In these verses, Allah SWT emphasizes that humans as stewards on earth have a responsibility to protect and preserve the environment so that it does not become damaged and deteriorated. “Eat and drink from the provision of Allah, and do not cause corruption and destruction on the earth.” (al-Baqarah: 60) “Various damages and calamities have arisen on land and sea because of what human hands have done; (these have arisen) because Allah wants to make them taste some of the consequences of the evil deeds they have done, so that they may return (to Him in repentance and awareness).” (al-Rum: 41) “Furthermore, in a hadith, the Prophet Muhammad (SAW) said...” “Furthermore, in a hadith, the Prophet Muhammad (SAW) said...” 1338 Corresponding Author:- Mohamad Zaidin Mohamad Address:- Research Institute for Islamic Products & Civilization (INSPIRE), Universiti Sultan Zainal Abidin, Gong Badak Campus, 21300 Kuala Nerus, Terengganu. 1338 Corresponding Author:- Mohamad Zaidin Mohamad Address:- Research Institute for Islamic Products & Civilization (INSPIRE), Universiti Sultan Zainal Abidin, Gong Badak Campus, 21300 Kuala Nerus, Terengganu. 1338 Corresponding Author:- Mohamad Zaidin Mohamad Address:- Research Institute for Islamic Products & Civilization (INSPIRE), Universiti Sultan Zainal Abidin, Gong Badak Campus, 21300 Kuala Nerus, Terengganu. Corresponding Author:- Mohamad Zaidin Mohamad Address:- Research Institute for Islamic Products & Civilization (INSPIRE), Universiti Sultan Zainal Abidin, Gong Badak Campus, 21300 Kuala Nerus, Terengganu. 1338 ISSN: 2320-5407 Green Technology in the Andalusia Era:- In its heyday, the Islamic government in Andalusia practiced several green technologies aimed at developing more efficient agriculture and water management. For example, a sophisticated and integrated irrigation system known as “aljibes,” which was a network of canals and reservoirs that collected, filtered, and distributed water throughout the Andalusia region. In addition, green technology in the field of architecture was also known in Andalusia, which used building design to reduce energy use and maximize natural air circulation. For example, buildings in Alhambra used a temperature regulation technique called “air conditioning” to maintain cool air temperature inside the building. Moreover, the Islamic government in Andalusia also developed sustainable agricultural practices, such as terracing systems to reduce soil erosion and maximize water usage, as well as using organic fertilizers and compost to increase soil fertility. Moreover, the Islamic government in Andalusia also developed sustainable agricultural practices, such as terracing systems to reduce soil erosion and maximize water usage, as well as using organic fertilizers and compost to increase soil fertility. However, green technology in Andalusia was not only applied by the government but also by the public. For example, the use of solar energy using concentrative mirrors (solar concentrators) used to heat water and generate steam in the textile and ceramic industries. Green technology in the era of Andalusia shows that the Islamic government in the past has practiced environmental principles and the use of sustainable and efficient natural resources. This indicates that these principles are not only relevant to the present but have also been applied since the past (Ruggles, 2008; Glick, 1992). In addition to green technology in agriculture and architecture, the Islamic government in Andalusia also applied green technology in the energy field. One of the most famous green technologies in the era of Andalusia was the “Almaden Tower” located in Toledo. This tower was used to generate electricity through wind power, where wind turbines on top of the tower produced electricity used to power machines. Moreover, the Islamic government in Andalusia also utilized other natural energy sources such as waterfalls and rivers to generate electricity through water turbines. The application of green technology in the era of Andalusia was also done in the field of waste processing, where the government built an integrated waste processing system using composting and aerobic composting methods to produce organic fertilizers. Int. J. Adv. Res. 11(05), 1338-1342 ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 11(05), 1338-1342 Int. J. Adv. Res. 11(05), 1338-1342 Int. J. Adv. Res. 11(05), 1338-1342 In the modern context, Muslims can develop green technology by utilizing available science and technology. This is in line with Islamic principles that encourage innovation and research in the field of green technology to preserve the environment and benefit humanity (Arthur, 2012). Green Technology in the Andalusia Era:- In conclusion, the Islamic government in Andalus practiced green technology in various fields, from agriculture, architecture, energy, to waste management. This green technology was implemented as part of sustainable and efficient environmental principles, which showed that thinking and acting to protect the environment has been an important concern in Islamic society for a long time (Caesarina& Aina, 2018; Hoffmann, 2014; Safran, 2021). Rasulullah SAW and Green Technology:- There are no specific historical records mentioning that Rasulullah SAWdirectly practiced green technology. However, the environmental principles and the use of natural resources described in the Quran and Hadith provide guidelines for Muslims to develop green technology in their daily lives. For example, in the Hadith narrated by Abu Dawud, it is mentioned that Prophet Muhammad taught his followers to pay attention to environmental cleanliness and health by disposing of waste properly. In addition, the Quran also explains the importance of maintaining ecosystem balance and the sustainability of living creatures on earth. In Islamic history, there were several figures who practiced environmental principles and the use of natural resources that aligned with green technology. For example, Caliph Umar bin Khattab, who was known for his attention to environmental cleanliness and improving the irrigation system in Medina. 1339 Conclusion:- Islam encourages its followers to behave in an environmentally friendly manner and to preserve the earth. In Islamic teachings, the earth is a trust from Allah that must be protected and maintained to remain sustainable. Green technology is environmentally friendly technology aimed at reducing the negative impact of humans on the environment. Islam teaches that humans should use knowledge and technology for the benefit of humanity and the environment, and not to destroy nature. Islam and green technology are not conflicting but complement each other. Islam teaches people to use technology wisely and responsibly, so that green technology can be a means for Muslims to preserve the earth and achieve happiness in this world and the hereafter. Follow-up Steps in Empowering Green Technology:- p p p g gy One of the steps that the Islamic world needs to take for technology to be more effective is to strengthen international cooperation. Here are some follow-up steps that the Islamic world can take to improve the effectiveness of green technology: One of the steps that the Islamic world needs to take for technology to be more effective is to strengthen international cooperation. Here are some follow-up steps that the Islamic world can take to improve the effectiveness of green technology: 1. Strengthen international cooperation: By strengthening international cooperation, Islamic countries can share knowledge, experience, and technology in the development of green technology. This can help accelerate the development of green technology and improve its effectiveness. 2. Increase funding for green technology: Increased funding from Islamic countries can help in the development of green technology and accelerate its use. In addition, funding can be used to raise awareness and education about green technology in the community. 3. Enhance collaboration between public and private sectors: Collaboration between public and private sectors can help accelerate the development of green technology and improve its effectiveness. For example, governments can provide incentives to private companies that develop green technology. 4. Raise public awareness: Raising public awareness about the importance of green technology can help increase its use and reduce negative impacts on the environment. This can be done through social campaigns and education in various media. 5. Increase research and development: Research and development can help improve the effectiveness of green technology and find new innovations in green technology development. Islamic countries can increase research and development through funding and international cooperation (Alshehri& Hussain, 2021; Islam, 2020; The World Bank, 2018). Int. J. Adv. Res. 11(05), 1338-1342 Int. J. Adv. Res. 11(05), 1338-1342 5. Saudi Arabia: Saudi Arabia has also begun taking steps to support green technology by launching the “Vision 2030” project aimed at reducing dependence on oil and developing renewable energy. In addition, they are building city parks and reducing the use of groundwater. 5. Saudi Arabia: Saudi Arabia has also begun taking steps to support green technology by launching the “Vision 2030” project aimed at reducing dependence on oil and developing renewable energy. In addition, they are building city parks and reducing the use of groundwater. Of course, there are many other Islamic governments that also support the development of green technology. This shows that more and more countries are beginning to realize the importance of protecting the environment and taking action to reduce negative impacts on the environment. The Islamic World Today and Green Technology:- The Islamic World Today and Green Technology:- y gy Several examples of Islamic countries that are serious about implementing green technology incl Several examples of Islamic countries that are serious about implementing green technology i Several examples of Islamic countries that are serious about implemen 1. United Arab Emirates (UAE): The UAE is one of the most active countries in the Middle East in developing green technology. They have built various green infrastructure, such as city parks and green lungs. In addition, they are also active in developing renewable energy technologies such as solar and wind energy and reducing the use of fossil fuels. 1. United Arab Emirates (UAE): The UAE is one of the most active countries in the Middle East in developing green technology. They have built various green infrastructure, such as city parks and green lungs. In addition, they are also active in developing renewable energy technologies such as solar and wind energy and reducing the use of fossil fuels. 2. Indonesia: The Indonesian government has also taken steps to support green technology. Some policies implemented include the development of biofuels and the promotion of renewable energy technologies such as solar and wind power. The government also provides incentives to reduce the use of single-use plastics. 3. Malaysia: The Malaysian government has taken steps to support green technology by launching the “Green Technology Financing Scheme” program to promote investment in green technology. Additionally, the government is also promoting the development of electric cars and solar power plants. 3. Malaysia: The Malaysian government has taken steps to support green technology by launching the “Green Technology Financing Scheme” program to promote investment in green technology. Additionally, the government is also promoting the development of electric cars and solar power plants. g p g p p p 4. Morocco: The Moroccan government has developed the “Morocco Solar Plan” program aimed at developing solar energy technology and reducing dependence on fossil fuels. They also developed the “Green Mosques” project to reduce the negative impact of mosques on the environment. 4. Morocco: The Moroccan government has developed the “Morocco Solar Plan” program aimed at developing solar energy technology and reducing dependence on fossil fuels. They also developed the “Green Mosques” project to reduce the negative impact of mosques on the environment. 1340 ISSN: 2320-5407 10. Mehran K. 2011. Innovation In Islam: Traditions and Contributions. Berkeley: University of Ca y 8. Hoffmann, R. (2014). An environmental history of medieval Europe. Cambridge University Pre References:- 1. Aburounia, H., & Sexton, M. (2006). Islam and sustainable development. 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https://openalex.org/W2766926036	https://repository.urosario.edu.co/bitstream/10336/23588/1/s13104-017-2840-1.pdf	English	null	A first description of the Colombian national registry for rare diseases	BMC research notes	2,017	cc-by	5,242	© The Author(s) 2017. 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 Objective: Orphan diseases must be considered a public health concern, underlying country-specific challenges for their accurate and opportune diagnosis, classification and management. Orphan disease registries have not yet been created in South America, a continent having a population of ~ 415 million inhabitants. In Colombia ~ 3 million of patients are affected by rare diseases. The aim of the present study was to establish the first Colombian national reg- istry for rare diseases. The registry was created after the establishment of laws promoting the development of clinical guidelines for diagnosis, management, census and registry of patients suffering rare diseases. Results: In total, 13,215 patients were recorded in the Colombian registry. The survey reported 653 rare diseases. The most common diseases were congenital factor VIII deficiency (hemophilia A) (8.5%), myasthenia gravis (6.4%), von Willebrand disease (5.9%), short stature due to growth hormone qualitative anomaly (4.2%), bronchopulmonary dysplasia (3.9%) and cystic fibrosis (3.2%). Although, a marked under-reporting of cases was observed, some patholo- gies displayed similar behavior to that reported by other initiatives and databases. The data currently available in the registry provides a baseline for improvement regarding local and regional surveys and the start for better understand- ing rare diseases in Colombia. Keywords: Rare disease registry, Orphan disease, Colombian health system © The Author(s) 2017. 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: paul.laissue@urosario.edu.co 1 Center For Research in Genetics and Genomics‑CIGGUR, GENIUROS Research Group, School of Medicine and Health Sciences, Universidad del Rosario, Carrera 24 No. 63C‑69, Bogotá, Colombia Full list of author information is available at the end of the article Mateus et al. BMC Res Notes (2017) 10:514 DOI 10.1186/s13104-017-2840-1 Mateus et al. BMC Res Notes (2017) 10:514 DOI 10.1186/s13104-017-2840-1 BMC Research Notes A first description of the Colombian national registry for rare diseases Heidi Eliana Mateus1, Ana María Pérez1, Martha Lucía Mesa2, Germán Escobar2, Jubby Marcela Gálvez1, José Ivo Montaño2, Martha Lucía Ospina3 and Paul Laissue1 Introduction to better understanding of their pathogenesis as well as contributing towards drug development and effec- tive therapeutic responses. Various national registries for rare diseases have been created (especially in North America and Europe) which could provide the basis for expanded international initiatives leading to more com- prehensive and homogeneous data being collected [6, 7]. For instance, countries having covering systematic collec- tions of data regarding specific rare diseases (or a group of diseases) would include France, Switzerland, Italy, Spain, the United Kingdom, Germany, the Netherlands, Norway, Sweden, Estonia and Poland [7, 8]. Further well-developed databases and initiatives for identifying patients affected by rare diseases include the Orphanet for rare diseases and orphan drugs, the Rare Disease- HUB (RD-HUB), the National Organization for Rare Dis- orders (NORD), the NIH/NCATS GRDR program and the Rare Diseases Clinical Research Network (RDCRN) [2, 9–11]. Orphan diseases represent a highly heterogeneous group of disorders which can affect any organ. Such pathologies involve a wide range of clinical features and severity. To date, 5000–8000 rare diseases have been recognized and most of them have a genetic origin [1, 2]. Rare diseases in the USA have been defined as those affecting less than 1 in 1250 individuals (1/2000 in the European Union) [1, 3, 4]. It has been estimated that 6–8% of the total popu- lation is affected by rare diseases, representing ~ 30 and ~ 25 million people in the European Union and the USA, respectively [5]. Rare disease patient registries are essential for collect- ing clinical and epidemiological data thereby leading Mateus et al. BMC Res Notes (2017) 10:514 Page 2 of 7 Page 2 of 7 Orphan disease registries have not yet been created in South America, a continent covering 14 countries and having an estimated population of 415 million inhabit- ants. Some associations and databases including specific pathologies have been created in Colombia and South American countries as the Colombian registry for Pompe disease and the LATAM fabry registry [12]. enrolled in Bogotá (32.65%) and the Antioquia region (14.33%). The Vaupés and Amazonas departments had the lowest percentage of patients reported in the registry (0.01 and 0.02%, respectively). Figure 1 represents preva- lence by region (Colombia is a Unitarian Republic, which is divided administratively and politically into 33 regions: 32 departments and Bogotá). The survey reported 653 rare diseases (~ 33% of all diseases included in the Colombian list of rare dis- eases). Establishing a national registry Detailed information on the steps for establishing the registry has been included as Additional files 1 and 2. Introduction Sixty-nine percent of these had a genetic origin. The most common diseases were congenital factor VIII deficiency (hemophilia A) (8.5%), myasthenia gravis (6.4%), Von Willebrand disease (5.9%), short stature due to growth hormone qualitative anomaly (4.2%), bronchopulmonary dysplasia (3.9%) and cystic fibro- sis (3.2%). The top 20 most common pathologies were identified in ~ 50% of reported cases. The most com- mon occurring pathologies in males were factor VIII deficiency (4.5%), short stature due to growth hormone qualitative anomaly (1.2%), bronchopulmonary dys- plasia (3.9%), myasthenia gravis (1.2%) and Von Wille- brand disease (1%). The top five rare diseases most commonly occurring in females were myasthenia gravis (8%), Von Willebrand disease (7.7%), diffuse cutaneous systemic sclerosis (4.7%), idiopathic and/or familial pul- monary arterial hypertension (3.5%) and bronchopul- monary dysplasia (3.4%) (Table 1). According to the International Statistical Classification of Diseases and Related Health Problems (ICD-10), the most preva- lent pathologies were diseases of the blood and blood However as yet, no structures for the systematic recording of rare diseases have been established. The present study provides a first description of a Colombian national registry for rare diseases. The main objective of the registry was to collect epidemiological data from patients affected by rare diseases and to propose distinct future national initiatives. These include the creation of diagnosis/treatment guidelines and the construction spe- cialized medical centers for rare diseases. Data corresponds to all living patients affected by rare diseases registered in the health care provider’s system (2015) Results BMC Res Notes (2017) 10:514 Page 3 of 7 Table 2 Distribution of rare diseases by nosologic group (ICD-10) Data corresponds to all living patients affected by rare diseases registered in the health care provider’s system (2015) ICD-10 n % Female Female% Male Male% 0–18 years % > 18 years % I 9 0.1 4 0.1 5 0.1 4 0.1 5 0.1 II 146 1.1 87 1.2 59 1.0 72 1.3 74 1 III 3455 26.1 1461 20.5 1994 32.8 1357 25 2098 26.9 IV 2040 15.4 1046 14.7 994 16.3 1222 22.5 818 10.5 V 128 1.0 66 0.9 62 1.0 52 1 76 1 VI 2120 16.0 1191 16.7 929 15.3 375 6.9 1745 22.4 VII 149 1.1 75 1.1 74 1.2 48 0.9 101 1.3 VIII 39 0.3 21 0.3 18 0.3 8 0.1 31 0.4 IX 478 3.6 298 4.2 180 3.0 92 1.7 386 5 X 15 0.1 8 0.1 7 0.1 8 0.1 7 0.1 XI 457 3.5 378 5.3 79 1.3 25 0.5 432 5.5 XII 512 3.9 292 4.1 220 3.6 120 2.2 392 5 XIII 987 7.5 792 11.1 195 3.2 122 2.2 865 11.1 XIV 32 0.2 20 0.3 12 0.2 17 0.3 15 0.2 XVI 546 4.1 261 3.7 285 4.7 536 9.9 10 0.1 XVII 2092 15.8 1130 15.8 962 15.8 1364 25.1 728 9.4 XVIII 3 0.0 2 0.0 1 0.0 3 0.1 0 0 XIX 7 0.1 0 0.0 7 0.1 4 0.1 3 0 Total 13,215 100.0 7132 100 6083 100 5429 41.1% 7786 58.9% Table 2 Distribution of rare diseases by nosologic group (ICD-10) s to all living patients affected by rare diseases registered in the health care provider’s system (2015) f International Statistical Classification of Diseases and Related Health Problems (ICD-10): Chapter I (certain infectious and parasitic diseases), Chapter II (neoplasms), Chapter III (diseases of the blood and blood-forming organs and certain disorders involving the immune mechanism), Chapter IV (endocrine, nutritional and metabolic diseases), Chapter V (mental and behavioural disorders), Chapter VI (diseases of the nervous system), Chapter VII (diseases of the eye and adnexa), Chapter VIII (diseases of the ear and mastoid process), Chapter IX (diseases of the circulatory system), Chapter X (diseases of the respiratory system), Chapter XI (diseases of the digestive system), Chapter XII (diseases of the skin and subcutaneous tissue), Chapter XIII (diseases of the musculoskeletal system and connective tissue), Chapter XIV (diseases of the genitourinary system), Chapter XV (pregnancy, childbirth and the puerperium), Chapter XVI (certain conditions originating in the perinatal period), Chapter XVII (congenital malformations, deformations and chromosomal abnormalities), Chapter XVIII (symptoms, signs and abnormal clinical and laboratory findings, not elsewhere classified), Chapter XIX (injury, poisoning and certain other consequences of external causes), Chapter XX (external causes of morbidity and mortality), Chapter XXI (factors influencing health status and contact with health services), Chapter XXII (codes for special purposes) forming organs (Chapter III) (26.1%), endocrine, nutri- tional and metabolic diseases (Chapter IV) (16%), con- genital malformations, deformations and chromosomal abnormalities (Chapter XVII) (15.8%), nutritional and metabolic diseases (15.4%) and diseases of the muscu- loskeletal system and connective tissues (Chapter XIII) (7.5%) (Table 2). Results In total, 13,215 patients were recorded in the Colombian registry: 54% (n = 7132) female and 46% (n = 6083) male. Population characteristics are summarized in Tables 1 and 2. Their mean age was 32 years old (SD 23.3; CI 95% 31.61–32.39) and 1199 (9.1%) individuals suffered disabil- ity on enrollment. The largest numbers of patients were Table 1 Most common disorders reordered in the Colombian registry for rare diseases Data corresponds to all living patients affected by rare diseases registered in the health care provider’s system (2015) Disease N % Prevalence per 100.000 n (Female) Prevalence per 100.000 n (Male) Prevalence per 100.000 Factor VIII deficiency 1117 8.5 2.37 77 0.32 1.040 4.47 Myasthenia gravis 839 6.4 1.78 570 2.39 269 1.16 Von Willebrand disease 779 5.9 1.65 551 2.31 228 0.98 Short stature due to growth hormone qualitative anomaly 559 4.2 1.19 203 0.85 356 1.53 Bronchopulmonary dysplasia 511 3.9 1.08 239 1.00 272 1.17 Cystic fibrosis 424 3.2 0.90 209 0.88 215 0.92 Diffuse cutaneous systemic sclerosis 408 3.1 0.87 331 1.39 77 0.33 Guillain–Barré syndrome 392 3.0 0.83 169 0.71 223 0.96 Idiopathic and/or familial pulmonary arte- rial hypertension 377 2.9 0.80 248 1.04 129 0.55 Acquired von Willebrand syndrome 281 2.1 0.60 211 0.88 0.00 Factor IX deficiency 270 2.0 0.57 73 0.31 197 0.85 Marfan syndrome 234 1.8 0.50 101 0.42 133 0.57 Acromegaly 232 1.8 0.49 134 0.56 98 0.42 Primary biliary cirrhosis 208 1.6 0.44 187 0.78 21 0.09 Multiple sclerosis-ichthyosis—factor VIII deficiency 196 1.5 0.42 147 0.62 49 0.21 on disorders reordered in the Colombian registry for rare diseases Table 1 Most common disorders reordered in the Colombian registry for rare diseases Mateus et al. Results The most commonly occurring dis- eases amongst patients aged under 18 years old were congenital malformations, deformations and chromo- somal abnormalities (25.1%), diseases of the blood and blood forming organs (25%), endocrine nutritional and metabolic diseases (22.5%), certain conditions origi- nated in the perinatal period (9.9%) and diseases of the eyes and adnexa (6.9%). The most commonly occurring pathologies in adults were diseases of the blood and blood forming organs (27%), diseases of the eye and adnexa (22.4%), diseases of the musculoskeletal system and connective tissue (11.1%), endocrine, nutritional and metabolic diseases (10.5%) and congenital malfor- mations, deformations and chromosomal abnormalities (9.4%) (Table 2). Data corresponds to all living patients affected by rare diseases registered in the health care provider’s system (2015) Discussion It should be noted that misdiagnosis frequently occurs in Colombia due to some rare dis- eases’ inherent complexity regarding their clinical and molecular diagnosis, together with few medical geneti- cists being available (about 60 all around the country). Such drawbacks surely merit accurate epidemiological follow-up and study regarding challenging, rare dis- eases. Underreporting is expected to be resolved in near future, at least in part, as it has been proposed that doc- tors should report rare diseases directly. to its inaccurate diagnosis and the unavailability of per- tinent treatment thereby leading to premature death [18]. The above, added to shortcomings related to inappropri- ate record-keeping procedures, might be the main vari- ables for its under-registry. Myasthenia gravis, a chronicle debilitating autoimmune muscular disorder, has 1.5–17.9 prevalence worldwide (7.7–37.7 in Europe) [19]. A first study of myasthenia gravis in Colombia’s Antioquia region revealed a 2.7 prevalence while the present study reported 1.78 cases per 100,000 habitants [20], suggesting doctors having good skills regarding its diagnosis. Interestingly, our group has found that, similarly to the report by Mazzucato et al. [21], some disorders displayed high prevalence during adulthood as haematological, musculoskeletal, eye and congenital diseases [21]. This might have been due to the fact that these pathologies, which frequently lead to disability (especially working limitations), are relatively well-diagnosed and treated. In a recent report, a novel strategy based on diagnostic codes, allowed the construction of a rare disease registry for patients from Western Australia [22]. This study was particularly interesting as it permitted to estimate the impact of rare diseases on the costs of discharges. Most neoplasias (representing around 5% of all of rare diseases in other countries) were also excluded from the present version of the Colombian registry because they are covered by specific legislation [23]. In other regis- tries neoplastic diseases prevalence ranges from 3.6 to 14.5% [21, 22]. It is worth stressing that several infec- tious diseases (e.g. tuberculosis, leishmaniasis) which are considered as rare in other countries, due to their low prevalence, frequently occur in Colombia since they have greater than 1/5000 prevalence and were thus excluded from the registry. Interestingly, for some groups of diseases, we have found differences between males and females. For instance, diseases of the blood and blood forming organs (Chapter III) affected 20.5 and 32.8% of women and males, respectively. This might have been due to the fact that common blood disorders (e.g. Discussion BMC Res Notes (2017) 10:514 Page 5 of 7 Although 5000–8000 (5833 in the Orphanet Database) rare diseases have been defined (1920 of which have been considered by Colombian legislation), our sur- vey only included 653 of them. This might have been due to the fact that many rare diseases involve multi- system organ damage/failure; this might lead to their inaccurate classification by doctors who have not been trained in identifying such kinds of disorder. Further- more, ICD-10, which was taken as the basis for classify- ing disorders in the present version of our registry, lacks numerous rare diseases (Table 2). Health systems have recently been recommended to use ORPHA codes, apart from ICD-10, for classifying rare diseases to improve the above [24]. Future ICD-11 is planned for use in 2018, promises an expanded repertoire of rare diseases which might improve the accuracy of identifying/classifying many disorders. It should be noted that misdiagnosis frequently occurs in Colombia due to some rare dis- eases’ inherent complexity regarding their clinical and molecular diagnosis, together with few medical geneti- cists being available (about 60 all around the country). Such drawbacks surely merit accurate epidemiological follow-up and study regarding challenging, rare dis- eases. Underreporting is expected to be resolved in near future, at least in part, as it has been proposed that doc- tors should report rare diseases directly. Although 5000–8000 (5833 in the Orphanet Database) rare diseases have been defined (1920 of which have been considered by Colombian legislation), our sur- vey only included 653 of them. This might have been due to the fact that many rare diseases involve multi- system organ damage/failure; this might lead to their inaccurate classification by doctors who have not been trained in identifying such kinds of disorder. Further- more, ICD-10, which was taken as the basis for classify- ing disorders in the present version of our registry, lacks numerous rare diseases (Table 2). Health systems have recently been recommended to use ORPHA codes, apart from ICD-10, for classifying rare diseases to improve the above [24]. Future ICD-11 is planned for use in 2018, promises an expanded repertoire of rare diseases which might improve the accuracy of identifying/classifying many disorders. Discussion It has been estimated that the prevalence of rare dis- eases is between 6–8% [13]. This value was estimated taking into account 5000–8000 disease while less than 2000 were included in the present version of the registry. Thus, a marked under-reporting of cases was observed in our study, since rare disease prevalence was 28/100,000 inhabitants (0.028%). This scenario highlights the fact that > 2.5 million patients have not been recorded or have not been diagnosed. Some of the most common diseases recorded in the registry’s present version (e.g. hemophilia A and myasthenia gravis) have been reported as having high prevalence in other databases (Orphanet) (Table 1). It has been reported that factor VIII deficiency preva- lence ranges between 12.8 ± 6 patients per 100.000 in high income countries while in other parts of the world it has been estimated at 6.6 ± 4.8 [14]. The present study showed that 1117 individuals were affected by factor VIII deficiency, having 4.5/100,000 prevalence similar to that (4.6) reported by the World Federation of Hemophilia for Colombia (2014) [15]. However, preliminary predictions had estimated that 5000 individuals should be affected by Mateus et al. BMC Res Notes (2017) 10:514 Page 4 of 7 Fig. 1 Prevalence of rare diseases in Colombia. Data corresponds to all living patients affected by rare diseases registered in the health care pro- vider’s system (2015) of rare diseases in Colombia. Data corresponds to all living patients affected by rare diseases registered in the health care pro 15) Fig. 1 Prevalence of rare diseases in Colombia. Data corresponds to all living patients affected by rare diseases registered in the health care pro- vider’s system (2015) this pathology in Colombia [16]. A preliminary national survey has already reported a higher number of cases (n = 1497) which pinpoints mistakes during their present recording. Indeed, a very rare disease, multiple sclerosis- ichthyosis-factor VIII deficiency (< 20 cases having been described worldwide to date) was reported as affecting 196 individuals in the present version of the registry [17]. This scenario suggests that these patients were probably affected by factor VIII deficiency alone. fi As previously stated, regarding countries having lim- ited resources, low hemophilia prevalence values in the present version of the registry might have been related Mateus et al. Discussion hae- mophilia A and B) are X-linked recessive diseases pre- dominantly affecting males. In the contrary, females are more commonly affected than males (11.1% vs 3.2%) by diseases of the musculoskeletal system and connective tissue (Chapter XIII) which includes rheumatoid arthritis and polyarthritis (Table 2). The data currently available in the registry, which is the first attempt to systematically record patients suf- fering rare diseases in Colombia, provides a baseline for improvement regarding local and regional surveys. This first approach for rare diseases registration will be con- tinued through a program called SIVIGILA (National Vigilance System of Public Health) created as a manda- tory tool for specialists and expertise centers to report patients with orphan diseases. This information must be the start for better understanding rare diseases in Colom- bia. It is clear that the data summarized in the present report has to be interpreted with caution as the reader must be aware that it has several limitations, especially due to many diseases having been inaccurately classified.h i The on-going and systematic recording of data by well- trained physicians should ensure the high quality (e.g. accuracy) of registered information in the future. Such scenario will be possible by proposing and implement- ing new educational programs for people working in the health field. Suitable codes (e.g. orphan-codes and/or OMIM codes) must also be used for identifying and mak- ing rare diseases traceable. These improvements should facilitate exchanges across registries and facilitate gov- ernment decisions leading to better health services for patients. Our group has observed that the Colombian regions most affected by under-registry (e.g. the islands of San Andrés and Providencia and the Arauca and Chocó departments) have traditionally been related with pov- erty. This data suggested that information regarding rare diseases across those Colombia’s vulnerable areas was due to population barriers to health access (Fig. 1). Mateus et al. BMC Res Notes (2017) 10:514 Page 6 of 7 References S h 1. Schieppati A, Henter J-I, Daina E, Aperia A. Why rare diseases are an important medical and social issue. Lancet (London, England). 2008;371:2039–41. 1. Schieppati A, Henter J-I, Daina E, Aperia A. Why rare diseases are an important medical and social issue. Lancet (London, England). 2008;371:2039–41. Consent for publication 17. Capra R, Mattioli F, Kalman B, Marcianò N, Berenzi A, Benetti A. Two sisters with multiple sclerosis, lamellar ichthyosis, beta thalassaemia minor and a deficiency of factor VIII. J Neurol. 1993;240:336–8. Authors’ contributions 8. Orphanet. Disease Registries in Europe. 2013.f HEM, AMP, MLM, GE, JMG, JIM, MLO, PL and HEM made substantive contribu- tions to conception and design, and analyzed the data. HEM and PL wrote the paper. All authors worked on the manuscript. All authors read and approved the final manuscript. 9. Richesson RL, Sutphen R, Shereff D, Krischer JP. The rare diseases clinical research network contact registry update: features and functionality. Contemp Clin Trials. 2012;33:647–56. 10. Groft SC, Rubinstein YR. New and evolving rare diseases research programs at the National Institutes of Health. Public Health Genomics. 2013;16:259–67. Additional files 2. Rubinstein YR, Groft SC, Bartek R, Brown K, Christensen RA, Collier E, et al. Creating a global rare disease patient registry linked to a rare diseases biorepository database: Rare Disease-HUB (RD-HUB). Contemp Clin Trials. 2010;31:394–404. Additional file 1: Figure S1. Methodology for the collection of data included in the registry. Additional file 1: Figure S1. Methodology for the collection of data included in the registry. Additional file 2. Additional methods include two sections: establishing a national registry and statistical analysis. Additional file 2. Additional methods include two sections: establishing a national registry and statistical analysis. 3. Taruscio D, Capozzoli F, Frank C. Rare diseases and orphan drugs. Ann dell’Istituto Super di sanità. 2011;47:83–93.ii 3. Taruscio D, Capozzoli F, Frank C. Rare diseases and orphan drugs. Ann dell’Istituto Super di sanità. 2011;47:83–93. 4. Kodra Y, Fantini B, Taruscio D. Classification and codification of rare dis- eases. J Clin Epidemiol. 2012;65:1026–7. 4. Kodra Y, Fantini B, Taruscio D. Classification and codification of rare dis- eases. J Clin Epidemiol. 2012;65:1026–7. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Although this study is the first initiative for systemati- cally record rare diseases in Colombia under-reporting has been largely observed. This was mainly due to doc- tors’ limited experience and knowledge regarding rare diseases and by the fact that severe forms of numerous conditions naturally leads to poor outcomes and short- term survival, despite suitable management. Received: 23 June 2017 Accepted: 23 October 2017 Received: 23 June 2017 Accepted: 23 October 2017 Abbreviations 5. Montserrat Moliner A, Waligóra J. The European Union policy in the field of rare diseases. Public Health Genom. 2013;16:268–77. 5. Montserrat Moliner A, Waligóra J. The European Union policy in the field of rare diseases. Public Health Genom. 2013;16:268–77. RD-HUB: Rare Disease-HUB; NORD: National Organization for Rare Disorders; RDCRN: Rare Diseases Clinical Research Network; CHSPM: Colombian Health and Social Protection Ministry’s; SISPRO: Colombian integral social protection information system; SIVIGILA: Public Health Surveillance System; SD: standard deviation; ICD: International Statistical Classification of Diseases. 6. Workshop on National Registries of RD. National Institute of Health. 2014. 6. Workshop on National Registries of RD. National Institute of Health. 2014. 7. Taruscio D, Vittozzi L, Choquet R, Heimdal K, Iskrov G, Kodra Y, et al. National registries of rare diseases in Europe: an overview of the current situation and experiences. Public Health Genomics. 2015;18:20–5. 7. Taruscio D, Vittozzi L, Choquet R, Heimdal K, Iskrov G, Kodra Y, et al. National registries of rare diseases in Europe: an overview of the current situation and experiences. Public Health Genomics. 2015;18:20–5. Ethics approval and consent to participate 18. Ferreira AA, Leite ICG, Bustamante-Teixeira MT, Guerra MR. Hemophilia A in Brazil—epidemiology and treatment developments. J Blood Med. 2014;5:175–84. The IRB of the Instituto Nacional de Salud, Bogotá, Colombia (INS) approved all of this study’s steps (Protocol: PRO-R02.55). For adults, as for participants younger than 16, the laws included in the resolution 1954 (2012) were followed concerning informed consents. Therefore, informed consent was not mandatory for the creation of the registry. Concerning personal data protection regulation, health insurers collected patients’ information regarding patients in line with law 1581/2012 which authorizes the use of personal information for statistical or scientific purposes. Authors received appropriate consent to access the data used to create the registry. 19. Boldingh MI, Maniaol AH, Brunborg C, Dekker L, Heldal AT, Lipka AF, et al. Geographical distribution of myasthenia gravis in northern Europe- results from a population-based study from two countries. Neuroepide- miology. 2015;44:221–31. 20. Sánchez JL, Uribe CS, Franco AF, Jiménez ME, Arcos-Burgos OM, Palacio LG. [Prevalence of myasthenia gravis in Antioquia, Colombia]. Rev Neurol. 34:1010–2. 20. Sánchez JL, Uribe CS, Franco AF, Jiménez ME, Arcos-Burgos OM, Palacio LG. [Prevalence of myasthenia gravis in Antioquia, Colombia]. Rev Neurol. 34:1010–2. 21. Mazzucato M, Visonà Dalla Pozza L, Manea S, Minichiello C, Facchin P. A population based registry as a source of health indicators for rare diseases: the 10-year experience of the Veneto Region’s rare diseases registry. Orphanet J Rare Dis. 2014;9:37. 21. Mazzucato M, Visonà Dalla Pozza L, Manea S, Minichiello C, Facchin P. A population based registry as a source of health indicators for rare diseases: the 10-year experience of the Veneto Region’s rare diseases registry. Orphanet J Rare Dis. 2014;9:37. Acknowledgements Not applicable. 13. Baldovino S, Moliner AM, Taruscio D, Daina E, Roccatello D. Rare diseases in Europe: from a wide to a local perspective. Isr Med Assoc J. 2016;18:359–63. 13. Baldovino S, Moliner AM, Taruscio D, Daina E, Roccatello D. Rare diseases in Europe: from a wide to a local perspective. Isr Med Assoc J. 2016;18:359–63. Competing interests Competing interests The authors declare that they have no competing interests. 14. Stonebraker JS, Bolton-Maggs PHB, Soucie JM, Walker I, Brooker M. A study of variations in the reported haemophilia A prevalence around the world. Haemophilia. 2010;16:20–32. 14. Stonebraker JS, Bolton-Maggs PHB, Soucie JM, Walker I, Brooker M. A study of variations in the reported haemophilia A prevalence around the world. Haemophilia. 2010;16:20–32. Availability of data and supporting materials section The datasets used and/or analysed during the current study are available fr Availability of data and supporting materials section Availability of data and supporting materials section The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. 15. Hemophilia WFo. Annual Global Survey. 2014.i The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. 16. Cortés Buelvas A. Hemofilia: terapéutica en las alteraciones hemostáticas. Colomb Med. 1999;30:10. 16. Cortés Buelvas A. Hemofilia: terapéutica en las alteraciones hemostáticas. Colomb Med. 1999;30:10. Author details 1 1 Center For Research in Genetics and Genomics‑CIGGUR, GENIUROS Research Group, School of Medicine and Health Sciences, Universidad del Rosario, Carrera 24 No. 63C‑69, Bogotá, Colombia. 2 Ministerio de Salud y Protección Social, Bogotá, Colombia. 3 Instituto Nacional de Salud, Bogotá, Colombia. 1 Center For Research in Genetics and Genomics‑CIGGUR, GENIUROS Research Group, School of Medicine and Health Sciences, Universidad del Rosario, 2 11. Rubinstein YR, McInnes P. NIH/NCATS/GRDR® Common Data Elements: a leading force for standardized data collection. Contemp Clin Trials. 2015;42:78–80. 11. Rubinstein YR, McInnes P. NIH/NCATS/GRDR® Common Data Elements: a leading force for standardized data collection. Contemp Clin Trials. 2015;42:78–80. Carrera 24 No. 63C‑69, Bogotá, Colombia. 2 Ministerio de Salud y Protección Social, Bogotá, Colombia. 3 Instituto Nacional de Salud, Bogotá, Colombia. 12. Villalobos J, Politei JM, Martins AM, Cabrera G, Amartino H, Lemay R, Ospina S, Ordoñez SS, Varas C. Fabry disease in latin America: data from the fabry registry. JIMD Rep. 2013;8:91–9. 12. Villalobos J, Politei JM, Martins AM, Cabrera G, Amartino H, Lemay R, Ospina S, Ordoñez SS, Varas C. Fabry disease in latin America: data from the fabry registry. JIMD Rep. 2013;8:91–9. facilitate inclusion of this topic in the National Rare Disease Plans. Public Health Genom. 2014;17:115–23. 24. Aymé S, Bellet B, Rath A. Rare diseases in ICD11: making rare diseases vis- ible in health information systems through appropriate coding. Orphanet J Rare Dis. 2015;10:35. Mateus et al. BMC Res Notes (2017) 10:514 Funding Thi t d This study was supported by the Universidad del Rosario (Grant CS/Genetics 017). 22. Walker CE, Mahede T, Davis G, Miller LJ, Girschik J, Brameld K, Sun W, Rath A, Aymé S, Zubrick SR, Baynam GS, Molster C, Dawkins HJS, Weeramanthri 22. Walker CE, Mahede T, Davis G, Miller LJ, Girschik J, Brameld K, Sun W, Rath A, Aymé S, Zubrick SR, Baynam GS, Molster C, Dawkins HJS, Weeramanthri Mateus et al. BMC Res Notes (2017) 10:514 Page 7 of 7 Page 7 of 7 Page 7 of 7 TS. The collective impact of rare diseases in Western Australia: an estimate using a population-based cohort. Genet Med. 2017;19:546–52. 23. Taruscio D, Arriola L, Baldi F, Barisic I, Bermejo-Sánchez E, Bianchi F, et al. European recommendations for primary prevention of congeni- tal anomalies: a joined effort of EUROCAT and EUROPLAN projects to • 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: TS. The collective impact of rare diseases in Western Australia: an estimate using a population-based cohort. Genet Med. 2017;19:546–52. 23. Taruscio D, Arriola L, Baldi F, Barisic I, Bermejo-Sánchez E, Bianchi F, et al. 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https://openalex.org/W3209917387	https://www.zdrav.tj/jour/article/download/258/250	Russian	null	Features of sexual behaviour of young people that determine the increase in the incidence of sexually transmitted infections (sti) in the Republic of Tajikistan	Zdravoohranenie Tadžikistana	2,021	cc-by	4,123	Н.Т. Курбанов Н.Т. Курбанов Здравоохранение Таджикистана, №3 (350), 2021 Здравоохранение Таджикистана, №3 (350), 2021 III 30 (47.6%), дараҷаи IV 13 (21.98%), дараҷаи V 2 (3.1%). Маълум шуд, ки аз 63 бемори марҳилаи 1. дар 26 (41,3%), II ст. - дар 16 (25,4%); Моддаи III - дар 13 (20,6%); IV. - дар 8 (12,7%), ва бемо- рони гирифтори Вст. - CKD вуҷуд надошт. VUR-и яктарафа дар 45 кӯдак (71,4%), VUR-и дутарафа дар 18 (28,5%) кӯдакон мушоҳида шудааст. Кор- рекцияи эндоскопии intravesical VUR бо усули зери пардаи луобӣ тазриқи «Dam +» гузаронида шуд. 19. Lee S.D., Jeong J.C., Chung J.M. Secondary sur- gery for vesicoureteral reflux after failed endoscopic in- jection: comparison to primary surgery. Investigative and Clinical Urology, 2016, No. 57, pp. 58-62. 20. Yılmaz İ., Peru H., Yılmaz F.H., Sekmenli T., Çiftçi İ., Kara F. Association of vesicoureteral reflux and renal scarringin urinary tract infections. Archivos Argentinos de Pediatría, 2018, Vol. 116, No. 4, pp. 542-547. ТАБОБАТИ РЕФЛЮКСИ ВЕЗИКОУРЕТЕРАЛИ ДАР КУДАКОН Хулоса. Ислоҳи эндоскопии VUR яке аз усулҳои муассиртарин ва осеби камтари табобат мебошад. Истифодаи маводи синтетикии био- логии мувофиқ «Дам +» дар ислоҳи рефлюкси везикоуретералӣ ба ноил шудан ба натиҷаҳои мус- бат мусоидат менамояд. Мақсади омӯзиш. Омӯзиши самаранокии ислоҳи эндоскопии рефлюси везикоуретералӣ дар кӯдакон бо истифода аз маводи синтетикии ҳаҷмбардор. Мавод ва усулҳои тадқиқот. Дар давраи аз соли 2016 то 2020 мо натиҷаҳои табобати 63 кӯдаки рефлюкси везикоуретералиро омӯхтем. Кӯдакон аз 6 моҳа то 17 сола буданд. Дараҷаи VUR: дараҷаи I (3.1%), дараҷаи II (25.3%), дараҷаи Калимаҳои калиди: дахолати эндоскопӣ, моддаҳои ҳаҷмӣ, рефлюкси везикоуретералӣ, кӯдакон. doi: 10.52888/0514-2515-2021-350-27-33 Vol. 25, No. 1, pp. 34-39. ХУЛОСА Натиҷаҳои тадқиқот ва муҳокима. Нати ҷаҳои тадқиқоти моро ҷамъбаст карда, бояд қайд кард, ки дар робита бо қатъ кардани сироятҳои рӯдаи пешоб, рафъи вайроншавии ихроҷи пешоб аз ҳолиб, беҳтар кардани вазифаи гурда пас аз ислоҳи VUR бо истифода аз Dam +натиҷаҳои мус- бат ба даст оварда шудаанд. Натиҷаҳои мусбии ислоҳи PMR 75.01%-ро ташкил медиҳанд. Х. Ибодов, Т.Ш. Икромов, Х.М. Мираков, К.М. Сайёдов, Р. Рофиев, С.К. Асадов 1. В переносном смысле – особенность в развитии, проявле- ние наивного подхода в отношениях, быту и в жизни. N.T. Kurbanov N.T. Kurbanov ГУ «НИИ профилактической медицины Таджикистана» МЗ и СЗНРТ Курбонов Назарали Талабович – соискатель ГУ «НИИ профилактической медицины в Таджи- кистана» МЗ и СЗНРТ, тел.: 907072013; «kurbonov-6969@mail.ru» ________________________________________________________________________________________ Цель исследования. Выявить особенности сексуального поведения молодежи, обуславливающие распро- странение инфекций, передающихся половым путем. Материалы и методы исследования. По специально разработанной анкете проведен опрос 500 лиц мужского и женского полов, относящихся к различным социальным слоям населения в возрасте 18-30 лет и находящихся в 4 районах республиканского подчинения. При этом социологический опрос основывался на типи- ческой выборке названной возрастной группы. Ответы на поставленные вопросы подвергли статистической обработке с использованием пакетов МS Office 2007 г. Результаты исследования и их обсуждение. Установлено, что соответствующая информированность респондентов об инфекциях, передающихся половым путем, и о мерах их профилактики остается на очень низком уровне. Более 21% из них имеют регулярные сексуальные контакты, причем как минимум каждый пятый не связывает с этим возможную реализацию риска заражения ИППП/ВИЧ/СПИДом. Среди ИППП наиболее часто встречаются гонококковая, трихомонадная, хламидийная, микроуреаплазмен- 27 Нигаҳдории тандурустии Тоҷикистон, №3 (350), 2021 ная инфекция, а также бактериальный вагиноз. Около 17% молодых людей считают, что в случае заражения этими болезнями за медицинской помощью обращаться будут только к своим знакомым врачам или средним медицинским работникам. ная инфекция, а также бактериальный вагиноз. Около 17% молодых людей считают, что в случае заражения этими болезнями за медицинской помощью обращаться будут только к своим знакомым врачам или средним медицинским работникам. Заключение. Доподлинно известно, что сегодня в молодежной среде приемлемы фактически все модели сексуального поведения и все типы сексуальных отношений. Однако, в Таджикистане существует некоторые особенности сексуального поведения молодежи (многоженство и развитие института гражданского брака). Установлен низкий уровень знаний и необходимость повышения осведомленности подростков и молодежи о негативных последствиях рискованного сексуального поведения. Ключевые слова: репродуктивное поведение, молодежь, образ жизни, факторы риска, инфекции, пере- дающиеся половым путем. FEATURES OF SEXUAL BEHAVIOUR OF YOUNG PEOPLE THAT DETERMINE THE INCREASE IN THE INCIDENCE OF SEXUALLY TRANSMITTED INFECTIONS (STI) IN THE REPUBLIC OF TAJIKISTAN SI «Research Institute of Preventive Medicine of Tajikistan» of Ministry of Health and Social Protection of the Population of the Republic of Tajikistan, Dushanbe, Tajikistan Kurbonov Nazarali Talabovich - research associate in the State Institution “Research Institute of Preventive Medicine in Tajikistan” of the Ministry of Health and Social Protection, tel.: 907072013, “Kurbonov-6969@mail.ru”. Aim. To identify the features of the sexual behavior of young people that cause the spread of sexually transmitted infections. Material and methods. A questionnaire was developed and a survey was conducted among 500 men and women that belong to various social groups of the population. The age of participants was 18 - 30 years and they were coming from 4 districts of republican subordination. The survey was based on a typical sample of the mentioned age group. Collected data were statistically processed using MS OFFICE 2007 packages. Results and discussion. It was found that the awareness of respondents about sexually transmitted infections and measures for their prevention remains at a very low level. More than 21.0% of respondents have active sexual life, and at least one in five does not associate this with the possible realization of the risk of contracting STIs / HIV / AIDS. About 17.0% of young people believe that if they become infected with these diseases, they will seek medical help only from their friends, physicians that they are familiar with, and paramedics. Conclusion. It is known for a fact that virtually all models of sexual behavior and all types of sexual relationships are present among young people today. However, in Tajikistan, there are certain features of the sexual behavior of young people (polygamy and civil marriage). A low level of knowledge and the need to raise awareness among adolescents and young people about the negative consequences of risky sexual behavior have been established. Keywords: reproductive behavior, youth, lifestyle, risk factors, sexually transmitted infections. путем (ИППП). путем (ИППП). Актуальность. Со второй половины ХХ века и до настоящего времени человечество сталкива- ется с массовой распространенностью инфекций, передающихся преимущественно половым путем, причем во множественном числе [2, 5, 8]. Так, среди молодых людей до 25 лет еже- годно происходит более 100 миллионов новых заражений, исключая ВИЧ, представляя глобаль- ную угрозу здоровью людей. Всего за названный период в мире было зарегистрировано 340 млн. новых случаев заболеваемости ИППП среди мо- лодых людей [1, 3, 6, 7]. Проблемы сексуального и репродуктивного здоровья подростков и моло- дежи, прежде всего, обусловлены низким уровнем репродуктивного образования, ранними браками, ростом гинекологической и венерической заболева- емости, беременностью и родами в юном возрасте, конфликтами и психологической напряженностью в семье [2, 6, 8]. В структуре факторов, формирующих здоро- вье молодежи особенно подросткового возраста, большое значение имеет такой фактор, как по- ведение, обусловленное растущим радикализмом и умственным «инфантилизмом»1 в некоторых странах. Особую актуальность поведенческий фак- тор приобрел в связи с ростом подверженности подростков инфекциями, передающимися половым Кроме того, социальная значимость определя- 28 Здравоохранение Таджикистана, №3 (350), 2021 ется снижением трудовой активности, супружеской неверностью, бесплодием, развитием психологи- ческого конфликта, домашним насилием и рас- падом семьи [1, 4, 5, 7]. Не исключено, что от уровня здоровья подростков зависит реализация их жизненных планов, стремление к социальному развитию, в т.ч. к созданию семьи и рождению детей, предопределяющих положение всей страны. Существенно возросли проблемы здорового образа жизни, сексуального и репродуктивного здоровья среди социально уязвимых групп на- селения, в т.ч. молодежи, что и определило цель настоящего исследования. Цель исследования. Выявить особенности сексуального поведения молодежи, обуславлива- ющие распространение инфекций, передающихся половым путем. Учитывая напряженную эпидемиологическую ситуацию, особенно в постсоветских странах, вы- званную распространением ИППП, руководство ВОЗ в рамках стратегии «Здоровье для всех в 21 веке» ещё в 1998 г. обратилось к правительствам стран, международным и неправительственным организациям с призывом объединить усилия в борьбе с названными заболеваниями. Материалы и методы исследования. По спе- циально адаптированной анкете с 1 октября по 15 ноября 2019 года проведен социологический опрос среди 500 молодых людей мужского и женско- го пола, которые относились к различным слоям гражданского общества, с различными уровнями образования, трудоустройства и семейного по- ложения, в 4 густонаселенных сельских районах республиканского подчинения: Рудаки, Вахдате, Гиссаре и Шахринау. Данная инициатива ВОЗ нашла поддержку среди большинства членов ООН, в т.ч. и в Респу- блике Таджикистан, где при содействии основных партнеров разрабатываются и реализуются Наци- ональные программы по профилактике и борьбе с социально значимыми заболеваниями на период до 2030 года. путем (ИППП). Из общего количества респондентов - 296 (59,2%) мужчин и 204 (40,8%) женщины, соот- ветственно женатыми оказались 122 (41,2%), за- мужними – 80 (39,2%) человек. При этом боль- шинство респондентов имели среднее, высшее, незаконченное высшее или среднее специальное образование. В настоящее время в нашей стране уже име- ются Национальные программы по борьбе с ту- беркулезом и ВИЧ/СПИД на период 2021-2025 г. Однако, к сожалению, в республике до сих пор нет Национальной программы по профилактике и борьбе с ИППП. Исследовалась типическая выборка лиц воз- растной категории от 18 до 30 лет. Анализ анкет- ных ответов проведен ретроспективным, клини- ко-статистическим и социально - аналитическим методом. Разработка и осуществление этой Програм- мы позволили бы в государственном масштабе реализовать комплекс необходимых лечебных и профилактических мероприятий в данном направ- лении. Поэтому необходимо ускорить работу по подготовке Национальной программы по профи- лактике и борьбе с ИППП на период до 2030 года. Результаты исследования и их обсужде- ние. Установлено, что из 134 студентов ВУЗов 25 (18,6%) человек имели опыт половой жизни, из 108 студентов ССУЗов - 16 (14,8%) человек; из 202 лиц имели половые связи до замужества или женитьбы - 64 (31,6%) человека (Рис 1.). Все 92 (18,4%) ученика средних школ опыта половой жизни не имели или на соответствующий вопрос не ответили. В обстановке мирового финансового кризиса, роста бедности, вынужденной внешней трудовой миграции наблюдается «взрыв» сексуальной актив- ности, обусловленной прежде всего социально- культурными факторами, уровнем воспитания в семье и школе. Рост общей заболеваемости подростков и мо- лодежи обусловлен такими социальными фактора- ми, как: низкая занятость, безработица и бедность, нехватка продуктов первой необходимости, недо- ступность медицинских услуг; рост преступности и бытового насилия; зависимость от азартных и компьютерных игр, наркотиков, низкая адаптация сельской молодежи к городским условиям жизни, нередко приводящими к ранней беременности, разводам, алкоголизму, проституции, табакоку- рению, росту инфекций, передающихся половым путем, и т.д. При этом обычными стали добрачные половые связи, аборты, рост подросткового материнства, заболеваний, предающихся половым путем, па- тологий репродуктивных органов [1, 4, 5]. Одна- ко в новых социально-экономических условиях широкое распространение ИППП характерно не для всей молодежи. Для определенной ее части конкретные условия жизни, уровень образования и воспитания зависят от характера усвоенных в процессе взросления норм поведения. 29 Нигаҳдории тандурустии Тоҷикистон, №3 (350), 2021 Рис. 1 Распределение респондентов по опыту половых связей (в обс. числах). Рис. 1 Распределение респондентов по опыту половых связей (в обс. числах). Факторами риска распространения ИППП в молодежной среде являются: ранее начало половой жизни, промискуитет и низкий уровень исполь- зования контрацепции. путем (ИППП). За последние 10 лет заболеваемость сифи- Самолечение и неполноценное лечение боль- 30 Здравоохранение Таджикистана, №3 (350), 2021 ных - наиболее частые причины роста заболевае- мости среди лиц активного репродуктивного воз- раста, значимость которых существенно растет, особенно в бедных и развивающихся странах. местностей, а также отсутствия мониторинга и оценки работы учреждений первичного звена здра- воохранения. К тому же сексуальность, как неотъемлемая часть полноценного человеческого бытия, остается исключительно личной и уникальной сферой пове- дения с многочисленными социально-культурными табу и правилами, что затрудняет ее открытое и свободное обсуждение. Установлено, что 16,8% респондентов в случае заражения ИППП не будут обращаться в лечебно- профилактические учреждения и, скорее всего, станут лечиться у знакомого врача или медсестры, что приводит к развитию хронических форм не- которых заболеваний. Самым фундаментальным барьером в Тад- жикистане, по нашему мнению, было и остается негласное табу на тему сексуальных отношений, которые воспроизводится в семье и сфере обра- зования. К развитию хронических форм заболеваний ИППП и факторов, обуславливающих тяжесть их течения, приводят несовершенство этапов оказания медицинской помощи, на первом месте из которых стоит профилактика. Для большинства людей сопротивление в от- ношении изучения полового поведения представ- ляют глубокосидящие страхи и сомнения в отно- шении их личной жизни. При этом, воссоздавая стыдливый образ, навязанный окружающими, мо- лодые люди упускают важную и содержательную информацию, касающуюся их репродуктивного здоровья. Из общего числа мужчин, в т.ч. студентов ВУЗов, в период летних каникул в 2018 и 2019 годах находились в трудовой миграции 65 (21,9%) человек, в том числе 30 (10,1%) из них имели слу- чайные половые связи. Этому способствует сексуальная активность молодых людей, практикующих разнообразие половых контактов со случайными партнерами, недостаточное использование контрацептивных средств, что объясняет значительную распростра- ненность среди них инфекционных заболеваний, передающихся половым путем. Более того, традиционно лица, заразившиеся ИППП, стараются скрыть факт заболевания от близких и друзей, так как боятся стигматизации и осуждения окружающих. Пропаганда основ здо- рового образа жизни, в т.ч. половое воспитание должно идти неразрывно с социальными процес- сами, которые происходят вокруг подростков и молодежи. Выявлено, что немногим более 29,0% сту- дентов высших и средних специальных учебных заведений имели информированность о механиз- ме передачи инфекций, передающихся половым путем. В 26,0% случаев респонденты мужского и женского полов указали на незнание ответов на вопросы об их индивидуальной профилактике. При этом средства контрацепции использовали всего 33,8% респондентов. Несмотря на разработку и внедрение в практи- ку работы образовательных учреждений различных программ профилактики заболеваний и повышения культуры здоровья, результативность их остается очень низкой. путем (ИППП). Употребление алкоголя и психотропных препаратов накладывают свой отпечаток на их сексуальное поведение. лисом и гонореей в Таджикистане снизилась до уровня показателей 3,3-3,5 на 100 тыс. населения, однако заболеваемость другими ИППП остается высокой, как среди взрослого населения, так и среди молодежи. Таким образом ИППП ложатся тяжелым бре- менем на бюджеты как домохозяйств, так и си- стемы здравоохранения в странах со средним и низким уровнями доходов, негативно влияя на общее благополучие и развитие людей. Опасность многих ИППП заключается в их скрытом течении, особенно в инкубационном и продромальном периодах, в результате возмож- ности широкого распространения. Установлено, что 29 (9,84%) лиц мужского пола прибегают к курению сигарет или насвая, среди девушек таких было всего 14 (6,9%). Ал- коголь употребляли 75 (25,34%) юношей и 16 (7,84%) девушек, причем в каждом четвертом случае - по праздникам и семейным торжествам. Следует напомнить, что наиболее часто встре- чаются такие ИППП: сифилис, гонорея, урогени- тальный хламидиоз, шанкроид, паховая гранулема (донованоз), хламидийная лимфогранулема, уроге- нитальный трихомониаз, герпетическая вирусная инфекция, анально-генитальные бородавки. Половым путем также передаются вирус им- мунодефицита человека (ВИЧ), вирус простого герпеса (ВПГ), вирус папиломы человека (ВПЧ), вирусы гепатитов В и С, а также некоторые пара- зитарные инфекции (фтириаз и чесотка), контаги- озный моллюск, амебиаз и другие. Среди учащихся старших классов, в т.ч. окончивших в них обучение, студентов ВУЗов и ССУЗов начало половой жизни совпадает с ис- полнением 18 лет в 4,1% случаев, 19 лет - в 5,0% случаев, 20 лет – в 6,1% случаев, 21 года- в 10,6% случаев и 22 лет - в 15,5% случаев. Как показыва- ют результаты исследования, респонденты раньше начинают половую жизнь, не всегда зная о методах контрацепции и часто не проходят регулярные обследования. Названные заболевания относятся к наиболее ярко выраженным «социальным болезням» и обыч- но имеют максимально широкое распространение в период экономической нестабильности, низкой занятости лиц активного трудового возраста, без- работицы, бедности. При этом 21,0% респондентов имели постоян- ного полового партнера (муж, жена и лица, состо- ящие в гражданском браке). В то же время 10,3% студентов учебных заведений перенесли ИППП, причем в 25,0% случаев заболевания не обраща- лись в лечебно-профилактические учреждения, занимаясь самолечением. Так же претерпевает изменение структура наиболее часто встречающихся ИППП за счет роста удельного веса заболеваний, которые чаще вызываются хламидиями и микоплазмами, чем гонококками. путем (ИППП). В связи с этим возникла необходи- мость расширения образовательных программ с использованиями инновационных технологий в работе по улучшению соответствующей осведом- ленности подростков и молодежи. Среди выше названных наиболее значимых проблем репродуктивного здоровья следует счи- тать основными сохранение в молодежной среде таких явлений, как раннее начало половой жизни и низкая санитарная культура. Высокие показатели заболеваемости ИППП в молодежной среде связа- ны с особенностями поведенческих характеристик подрастающего поколения, особенно в условиях трудовой миграции. Таким образом, необходима работа по моти- вированности молодежи к сохранению репродук- тивного здоровья путем улучшения пропаганды здорового образа жизни в школах, высших и средних специальных учебных заведениях, что позволит решить демографические, социальные и медицинские проблемы репродуктивного здоровья молодежи в целом. 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Беш аз 21,0% онҳо мунтазам ба муносибатҳои шаҳвонӣ машғул буда, ҳадди ақал ҳар шахси 5-ум хатари имконпазири сироятёбӣ ба ББРҶИ, ВНМО- ро алоқаманд намемешуморад, тақрибан 17,0% ҷавонон чунин ақида доранд, ки ҳангоми мубтало ба ин бемориҳо танҳо ба табибони шиноси худ, ё кормандони миёнаи тиббӣ муроҷиат ҳоҳанд кард. Калима ва ибораҳои асосӣ: рафтори тазза курӣ (репродуктивӣ), ҷавонон, тарзи ҳаёт, омилҳои хатар, сироят, тавассути муносибатҳои ҷинси интиқолёбанда. doi: 10.52888/0514-2515-2021-350-33-40 УДК 616-053.2;615.37 Г.С. Мамаджанова1, З.К. Умарова2, Г.М.Саидмурадова3 ВАКЦИНАЦИНАЦИЯ ПЕНТИВАЛЕНТНОЙ АКДС И АДС ВАКЦИНОЙ ЗДОРОВЫХ И ЧАСТО БОЛЕЮЩИХ ДЕТЕЙ 1Кафедра детских болезней № 1, ГОУ «ТГМУ им. Абуали ибни Сино» 2Кафедра семейной медицины № 2, ГОУ «ТГМУ им. Абуали ибни Сино» 3Кафедра детских инфекций, ГОУ «ТГМУ им. Абуали ибни Сино» Мамаджанова Гульнора Сидикжановна – к.м.н., доцент, заведующая кафедрой детских болез- ней №1 ГОУ «Таджикский государственный медицинский университет им. Абуали ибни Сино»; Тел.: +(992)918720903; E-mail: gulnora.mamadjanova@gmail.com Цель исследования. Изучить вакцинированных здоровых и часто болеющих детей на формирование про- тиводифтерийного иммунитета. Материалы и методы исследования. Под нашим наблюдением было 50 практически здоровых и 92 часто болеющих детей. Всем детям проводилась вакцинация трехкратно пентавалентной вакциной, а также ревак- цинация АКДС и АДС. Определяли формирование специфического противодифтерийного иммунитета через 6 месяцев, 1 год и 5 лет. Интерпретировали результат по окончательному значению оптической плотности уровни защиты: базовая -0,01 до 0,1; полная защита - >0,1; долгосрочная защита - >1,0. Результаты исследования их обсуждение. Через 6 месяцев напряжённость поствакцинального имму- нитета как у здоровых, так и у больных детей составили высокий процент: полная (>0,1) (45,8%) и базовая защита (0,01 до 0,1) (31,0%). Статистическая значимость снизилась через год: полная защита (45,8% против 12,1%, p<0,001), и повысилась отсутствие защиты (2,6% против 55,7%, p<0,001), что указывало на выработку нестойкого иммунитета в связи, с чем была проведена ревакцинация АКДС и АДС. Через 5 лет после ревак- цинации, специфический иммунитет имел тенденцию к нарастанию как у здоровых детей, так и у больных. Заключение. Для создания стойкого и продолжительного иммунитета при использовании «убитых» вакцин и анатоксинов необходимы ревакцинирующие прививки через различные сроки после курса вакцинации часто болеющим детям, на что указывают данные базовой и полной защиты. Ключевые слова: дети, дифтерия, специфический иммунитет, 33
https://openalex.org/W3194817536	https://ela.kpi.ua/bitstream/123456789/47364/1/madt_2021-1_p79-88.pdf	Russian	null	Application of Avinit vacuum plasma technologies Avinit to the manufacture of high-precision full-size gears	Mechanics and advanced technologies	2,021	cc-by	6,663	Received: 20 February 2021 / Accepted: 24 May 2021 All parameters of the ring gear manu- factured using the Avinit duplex technology correspond to accuracy grade 4 in accordance with the requirements of technical documentation. The gears manufactured using the Avinit duplex technology were tested as part of the AI-450M engine reducer at the Ivchenko-Progress hydraulic brake stand according to the program of equivalent cyclic tests. A pair of experimental gears were installed in the engine reducer instead of the serial wheels of the second stage of the reducer. The total test time of the wheels was 26 hours. After testing, no damage to the gear, including the Avinit coating, was found. Antifriction coating Avinit C310 with a thickness of 1.5 microns does not distort the geometry of the tooth profiles during testing as part of the AI-450M engine reducer. Measurement of the parameters of the teeth showed a complete absence of wear. Keywords: development of Avinit duplex technologies, properties of the nitrided layer, coating Avinit characteristics, bench tests of high-precision gears manufactured using Avinit duplex technology. Mech. Adv. Technol. Vol. 5, No.1, 2021, рр. 79–88 Mech. Adv. Technol. Vol. 5, No.1, 2021, рр. 79–88 DOI: 10.20535/2521-1943.2021.5.1.234484 UDC 621.9.031 Received: 20 February 2021 / Accepted: 24 May 2021 Received: 20 February 2021 / Accepted: 24 May 2021 stract. Avinit duplex technologies have been developed, combining Avinit N plasma nitriding of finished high-precision parts w plication of Avinit superhard antifriction coatings in a single technological process pp f p f g g g p Due to the absence of a brittle layer on the nitrided surface after precision nitriding, the preservation of the original geometric dimensions that do not require further mechanical refinement, and the compatibility of the processes of plasma precision nitriding of Avinit N and the vacuum plasma depo- sition of functional coatings Avinit C, duplex technologies allow the deposition of strong adhered, high-quality coatings. The effect of the duplex process on the dimensions of parts during plasma nitriding of high-precision gears and the application of Avinit C functional coatings was investigated, the properties of the nitrided layer and the parameters of Avinit coatings were studied. Plasma precision technology Avinit N allows nitriding of finished parts without changing dimensions, including gears of 4 degrees of accuracy. Avinit N nitriding time is 2 ... 4 times less than with gas nitriding. g g g e coating of Avinit C310 parts increases the microhardness of the surfaces of the parts and reduces the coefficient of friction, while hesion to the working surfaces of the gear teeth and bearing raceways. g f f g g y ing of high-precision gears with accuracy grade 4 using Avinit duplex technologies was carried out. Manufacturing of high-precision gears with accuracy grade 4 using Avinit duplex technologies was carried out. ts shows that, within the measurement accuracy, no changes in the profiles of the teeth, their location on the ring gear, as well as ear ring relative to the measuring bases are observed. Plasma nitriding makes it possible to reduce the nitriding time by more than two times compared to gas nitriding, while the thickness of the layer of the brittle phase with the maximum surface hardness is ensured within the specified values to ensure the necessary indicators of contact and bending long-term strength in the manufacture of gears according to the degree of accuracy 4 without grinding after nitriding. Measurements of the ring gear after nitriding and coating showed that there were no changes in the geometry of the gear processed by duplex tech- nology. Avinit C310 anti-friction coating 1.5 microns thick does not distort the geometry of the tooth profiles. Application of Avinit vacuum plasma technologies Avinit to the manufacture of high-precision full-size gears A. Sagalovych1 • V. Popov1 • V. Sagalovych1 • S. Dudnik1 • A. Edinovych2 A. Sagalovych1 • V. Popov1 • V. Sagalovych1 • S. Dudnik1 • A. Edinovych2 ISSN 2521-1943 Mechanics and Advanced Technologies 1 JSC “FED”, Kharkiv, Ukraine 2 SE “Ivchenko Progress” Zaporizhya, Ukraine 1. The use of plasma precision nitriding Avinit N for the manufacture of high-precision gears Diffusion saturation of the steel surface with nitro- gen occurs at a temperature of about 550 °C. which en- sures the absence of phase transformations in steel, be- cause the process takes place at a temperature below the temperature of low tempering after quenching. This sig- nificantly reduces the curvature and, consequently, the allowance removed during grinding. Works [11, 12] are devoted to the study of the pos- sibility of using plasma precision nitriding Avinit N for the manufacture of finally finished high-precision gears with a degree of accuracy 5...4 without grinding after ni- triding, the study of the effect of plasma nitriding on the size of parts in plasma nitriding gears nitrided layer, and the study of plasma precision nitriding Avinit N high- precision gears. The disadvantages of traditional industrial technol- ogies for strengthening the surfaces of steel parts by the formation of metal nitrides in the surface layer are the long duration of the process (2 ... 3 times longer than during cementation) and the small depth of the diffusion layer [2]. In order to study the properties of plasma nitriding, a gear with a degree of accuracy [11] was made. The gear wheel is made of steel 34NiCrMoV14-5 with the following heat treatment before nitriding opera- tion: Increasing the saturation rate is achieved by ionic nitriding in a dilute nitrogen atomic medium of the glow discharge, which reduces the process time by 1.5...2 times. – quenching – 900 °C (3.5 h); In works [3, 4] the possibility of manufacturing gears with a degree of accuracy from 6 to 5 without grind- ing after ionic nitriding is shown. – leave – 620 °C (6 hours); – vstabilizing leave after machining – 550°C (6 hours). There are no data on the use of nitriding in the manufacture of more loaded gears, where it is necessary to use accuracy degrees 5...4 [5–7]. Plasma nitriding was performed on a vacuum- plasma cluster Avinit [19]. The part was completely nitrided, and the degree of nitrogen saturation depended on the plasma concentration in a particular area of the part. In [8–10] it was shown that even greater accelera- tion of the process than with ionic nitriding in a glow dis- charge occurs during plasma nitriding. To determine the effect of nitriding on the geomet- ric dimensions of the experimental part was measured before and after nitriding. The gear ring gear was meas- ured. Introduction gears and rolling bearings, which is determined by the properties of the materials from which they are made, in particular, the hardness of the material core, hardness of the working hardened surface, depth of the strengthened layer, surface roughness, coefficient of friction [1]. At creation of new materials with ultrahigh charac- teristics technologies of modification of surface layers of contacting materials and drawing functional coverings are more and more successfully used. Typically, standard manufacturing technologies for many industrial parts designed to work in high-precision gears, hinges and plain bearings include pre-cementation, subsequent grinding and polishing of work surfaces to achieve the required geometric dimensions. A very important characteristic of structural materi- als is the long fatigue strength of the working surfaces of  A. V. Sagalovych avinit @avinit.ua  A. V. Sagalovych avinit @avinit.ua Nitriding is much less commonly used as a method of strengthening machine parts and tools to provide func- tional properties. ISSN 2521-1943 Mechanics and Advanced Technologies © The Author(s). The article is distributed under the terms of the license CC BY 4.0. 80 Mech. Adv. Technol. Vol. 5, No. 1, 2021 Comparative tribotechnical tests for contact fatigue strength during rolling friction with sliding of surfaces reinforced with cementation and plasma nitriding Avinit N with contact loads σmax ≈ 1000 MPa were carried out in [13]. The main disadvantages of traditional industrial ni- triding technologies (gas furnace nitriding, liquid nitrid- ing, etc.) are the presence of a solid brittle ε-phase on the nitrided surface and significant distortion of parts, which further grinding and polishing of work surfaces signifi- cantly reduces the contact strength of the surface and in- creases manufacturing complexity products. The results of tests based on 1,000,000 cycles with contact loads σmax = 1140 MPa, typical for medium- loaded surfaces, showed that the integrated multi-cycle resistance to fatigue wear (destruction) of samples rein- forced with nitriding Avinit N with a layer depth of 0.25 mm, more than 10 times higher than samples rein- forced by cementation with a layer depth of 1.2 mm. Nevertheless, nitriding processes to increase con- tact endurance have significant prospects in comparison with traditionally used cementation processes in terms of increasing surface hardness, especially by eliminating machining after nitriding. A significant increase in contact endurance pro- vides a reduction in the coefficient of friction of the con- tact surfaces. Introduction At the end of the twentieth century, the level of sur- face hardness for cemented and nitrided parts stabilized at about 60 HRC, but if for the former it was actually the limit, for the latter it was possible to increase hardness and, accordingly, contact endurance by eliminating ma- chining after nitriding. 1. The use of plasma precision nitriding Avinit N for the manufacture of high-precision gears We have developed the process of plasma precision nitriding Avinit N [8–10], based on the diffusion satura- tion of the surface layer of steel parts with nitrogen in high-density (n ≥ 1010–1011 см–3) low-temperature very nonequilibrium plasma of nitrogen and argon. significant- ly reduces the duration of nitriding, ensures the absence of deformation (curvature) of the parts while maintaining the original geometric dimensions after nitriding with an ac- curacy of 1–2 μm, which avoids finishing grinding of parts and get the operation of precision nitriding “in size”. The geometric dimensions of the gear ring sample were measured on the same control and measuring ma- chine “WENZEL” with a measurement error of 0.8 μm before and after nitriding under the same conditions, on the same teeth. The measurement results are given in table 1. The analysis of the results shows that within the ac- curacy of measuring any changes in the profiles of the teeth, their location on the toothed crown, as well as the location of the toothed crown relative to the measuring bases is not observed. Studies [11, 12] have shown that the methods of plasma precision nitriding Avinit N have a high efficiency for the manufacture of final finished high-precision gears with a degree of accuracy of 5...4 without grinding after nitriding. 81 Mech. Adv. Technol. Vol. 5, No. 1, 2021 Table 1. Measurements of the toothed crown. Table 1. Measurements of the toothed crown. Name parameter Condition ally marking Deviation of parameters, мкм Deviation limit to nitriding after nitriding Tooth number Tooth number 5th degree 1 23 43 66 1 23 43 66 Tooth profile error Fα 4 5 3 3 5 5 4 3 8 Tooth line error Fβ 2 3 2 4 3 3 4 4 8,5 Accumulated step error FP 20 20 19 Borderline step deviation γP ±4 ±4 ±7 The measurement of the hardness of the nitrided layer, starting from the edge, was performed with a dis- creteness of 0.05 mm to a depth of 0.5 mm (table 2). It was established [12], that the plasma precision nitriding of Avinit N ensured the production of gears with a degree of accuracy of 5 without grinding after nitriding. Changes in the geometry after plasma high-precision ni- triding Avinit N is not observed, and all parameters of the ring gear meet the accuracy of 5 according to the re- quirements of the technical documentation. 1. The use of plasma precision nitriding Avinit N for the manufacture of high-precision gears When nitrid- ing, the parameters of the teeth of the gears are stably maintained within the accuracy of 5. Some change in the profile was observed at the tops of the teeth, which is due to the increased electric potential in this area. Measure- ment of the part after plasma nitriding did not reveal a similar phenomenon, on the contrary, on the tops of the teeth there is a slight decrease in hardness, which, howev- er, should have a positive effect on the performance of the gears. Table 2. The results of hardness measurements № Distance from the edge, mm Hardness, Нµ 1 0,025 709,5 2 0,05 712,5 3 0,10 679 4 0,15 606 5 0,20 565 6 0,25 466,5 7 0,30 434,5 8 0,35 434,5 9 0,40 431,5 Table 2. The results of hardness measurements In order to study the properties of plasma nitriding of gears, a technological sample based on a gear made of 5 degrees of accuracy was used, on which nitriding modes were tested and which was subjected to geometry meas- urements and metallurgical research [12]. On the technological sample made of the investi- gated gear after nitriding and measurements, metallurgical researches on definition of hardness, thickness of the ni- trided layer, distribution of microhardness on a layer, mi- crostructure of a layer and a core were carried out. Cities for measuring the hardness of the readings in Fig. 2. Microstructural analysis showed that the depth of the nitrided layer is ~ 0.3 mm, which meets the require- ments of the technical documentation (0.5...0.7 mm initial minus 0.15 mm allowance for grinding), but with greater surface hardness (more than 1000 HV). The appearance of the technological sample (frag- ment of the gear) is shown in Fig. 1. Fig. 1. Fragment of the gear X-ray diffraction analysis showed that the ε-Fe3N phase was not determined in the nitrided layer, and in- tense reflections of the a-Fe phase and very weak reflec- tions of FeC carbide were detected (Fig. 3). Plasma nitriding reduces the time required for ni- trogen diffusion by more than twice compared to gas ni- triding, while the thickness of the layer of brittle phase with maximum surface hardness is provided within the specified values to ensure the required indicators of con- tact and bending strength. Fig. 1. Fragment of the gear 82 Mech. Adv. Technol. Vol. 5, No. 1. The use of plasma precision nitriding Avinit N for the manufacture of high-precision gears 1, 2021 JSC “FED” (Kharkiv, Ukraine) and STC “Nano- technology” (Kharkiv, Ukraine) have developed complex coating technologies Avinit that combine in one cycle such methods as plasma-chemical, vacuum-plasma and others, which allow to obtain coatings from almost any elements, alloys, ceramics and other materials [14–16]. These technologies allow to perform multilayer antifric- tion coatings, for example, Avinit C310 [17, 18], which is a combination of nanolayers of titanium nitrides, alumi- num nitrides and titanium. This coating has a unique coef- ficient of dry friction. The advantage of these technolo- gies is also the ability to control the amount of heating of the part, which is mandatory with limited heat resistance of the coated materials. The developed technologies for applying Avinit multifunctional coatings have shown the high antifriction properties of Avinit coatings and high efficiency in many industrial applications when applied to critical parts in aggregate construction. transport engi- neering and power engineering [19, 20]. a b Fig. 2. Imprints of microhardness measurement – a (the section is not etched), – b (the section is etched) a a a b However, obtaining high-quality Avinit coatings on surfaces nitrided by traditional methods is associated with great difficulties, mainly due to the presence on the nitrid- ed surface of a fairly thick layer of brittle ε - phase and significant distortion of parts. To bring the dimensions to the requirements of the technical documentation after nitriding requires further grinding and polishing of work surfaces, which significantly reduces the contact strength of the surface and increases the complexity of manufac- turing products. The mechanical parameters of this thick brittle layer are poorly compatible with the coating. and as it turned out, further vacuum-plasma application of high- quality wear-resistant coatings is possible only with the complete removal of these fairly thick layers from the coated work surfaces. For complex surfaces, this becomes quite difficult and time-consuming, and sometimes simply unsolvable technical problem. b Fig. 2. Imprints of microhardness measurement – a (the section is not etched), – b (the section is etched) Fig. 3. Results of X-ray diffraction analysis of nitrided steel Our research has shown that the absence of a brittle surface layer after plasma nitriding allows further applica- tion of high-quality functional coatings Avinit without any mechanical improvements of the nitrided surface. 1. The use of plasma precision nitriding Avinit N for the manufacture of high-precision gears In this regard, it is of undoubted interest to develop duplex technology that combines plasma nitriding of fin- ished high-precision parts with subsequent application of superhard antifriction coatings Avinit in a single process, which will avoid many difficulties associated with the use of gas nitriding technology used in the production of details. Fig. 3. Results of X-ray diffraction analysis of nitrided steel 2. Development of duplex technologies (plasma precision nitriding Avinit N + hard coating Avinit C) 2. Development of duplex technologies (plasma precision nitriding Avinit N + hard coating Avinit C) Due to the absence of a brittle layer on the nitrided surface after precision nitriding, preservation of the origi- nal geometric dimensions that do not require further me- chanical refinement, and compatibility of the processes of plasma precision nitriding Avinit N and vacuum-plasma application of functional coatings, Avinit duplex this sig- nificantly improves the economic performance of the manufacture of parts. Even more significant results in increasing the con- tact long-term strength can be expected when applying antifriction coatings to the nitrided surface. When using metal surface coatings (copper, palla- dium), such antifriction coatings have limited perfor- mance due to abrasive wear. In the 1990s, tungsten car- bide-based ceramic hard coatings began to be introduced in the West. Duplex processes, including plasma nitriding and subsequent vacuum-plasma application of wear-resistant 83 Mech. Adv. Technol. Vol. 5, No. 1, 2021 coatings in a single process, are carried out on a vacuum- plasma cluster Avinit. Changing the process parameters (pressure, tem- perature, time, discharge voltage) allows to provide the specified properties of nitrided surfaces. Reduction of nitriding time allows to make details of the increased ac- curacy. First, plasma precision nitriding of Avinit N parts [8, 9].is performed. Process parameters and its duration (usually T ∼ 500…550 °C, t ∼ 2…4 h) are determined based on the results of previous experiments. By means of the corresponding electronic switching the Avinit installation is switched to a coating mode. a Number N Al Si Ti Cr Fe Total, % 006 4,02 36,56 59,42 100 007 2,69 20,45 76,87 100 008 2,00 7,05 90,94 100 009 2,12 4,16 93,72 100 010 1,41 2,92 86,81 1,23 7,63 100 011 1,08 54,8 5,75 38,37 100 012 0,27 16,47 9,85 73,41 100 013 0,37 3,67 10,66 85,3 100 b Fig. 4. The appearance of the coating Avinit C310 on the sample with the specified places of analy- sis. – a, the approximate chemical composition of the analyzed places – b The temperature of the parts is reduced to the pa- rameters required for vacuum-plasma coating of Avinit C (T ∼ 200…300 °C, t ∼ 1…2 h) according to the estab- lished technology [17, 18]. Coating on the witness samples is carried out in conjunction with the working parts. 2. Development of duplex technologies (plasma precision nitriding Avinit N + hard coating Avinit C) After the coating process, a careful control is car- ried out on the compliance of the thickness and micro- hardness of the applied coatings to the requirements of the technical documentation by metallographic and profilo- graphic analyzes of the sections of the witness samples. a a Number N Al Si Ti Cr Fe Total, % 006 4,02 36,56 59,42 100 007 2,69 20,45 76,87 100 008 2,00 7,05 90,94 100 009 2,12 4,16 93,72 100 010 1,41 2,92 86,81 1,23 7,63 100 011 1,08 54,8 5,75 38,37 100 012 0,27 16,47 9,85 73,41 100 013 0,37 3,67 10,66 85,3 100 b Changing the process parameters (pressure, tempera- ture, time, discharge voltage) allows to provide the speci- fied properties of nitrided surfaces. Reduction of nitriding time allows to make details of the increased accuracy. On the JEOL 6360LA electron microscope, the Avinit coating on the surface of the nitrided layer was studied by the PCMA method. The research results are presented in Fig. 4–6. On the surface of the part the presence of titanium, aluminum, nitrogen, which are absent in the part made of steel 34NiCrMoV14-5, but present in the coating Avinit C310, which is a multilayer composition of layers of tita- nium, titanium nitride and aluminum nitride. The thickness of the coating is 1.5 μm. Fig. 4. The appearance of the coating Avinit C310 on the sample with the specified places of analy- sis. – a, the approximate chemical composition of the analyzed places – b Fig. 4. The appearance of the coating Avinit C310 on the sample with the specified places of analy- sis. – a, the approximate chemical composition of the analyzed places – b Fig. 4. The appearance of the coating Avinit C310 on the sample with the specified places of analy- sis. – a, the approximate chemical composition of the analyzed places – b The microhardness of the coating was 2500...3000 HV. Developed Avinit duplex technologies, including plasma nitriding Avinit N and ion-plasma application of antifriction wear-resistant coatings Avinit C were applied to the manufacture of full-size high-precision gear in Fig. 7. Fig. 5. Appearance of the Avinit C310 coating on the sample in mapping mode Fig. 5. Appearance of the Avinit C310 coating on the sample in mapping mode 84 Mech. Adv. Technol. Vol. 5, No. 1, 2021 Fig. 6. Appearance of the Avinit C310 coating on the sample in line analysis mode. 2. Development of duplex technologies (plasma precision nitriding Avinit N + hard coating Avinit C) The thickness of the coating is 1.5 μm Fig. 6. Appearance of the Avinit C310 coating on the sample in line analysis mode. The thickness of the coating is 1.5 μm Fig. 8. Appearance of the witness sample Fig. 7. Full-size high-precision gear, made using duplex technology Avinit (nitriding Avinit N + coating Avinit C310) Fig. 8. Appearance of the witness sample The results of studies of plasma nitriding of a gear wheel with plasma nitriding and Avinit C310 coating are described in [11]. Fig. 7. Full-size high-precision gear, made using duplex technology Avinit (nitriding Avinit N + coating Avinit C310) Measurement of the hardness of the nitrided layer, starting from the edge of the tooth, was performed with a resolution of 0.05 mm to a depth of 0.5 mm. After carrying out work on testing the nitriding of the gear, made with 5 degrees of accuracy, was made ex- perimental full-size high-precision helical gear [21] with a degree of accuracy of 4 for plasma nitriding, followed by application of antifriction coating Avinit C310. The hardness of the nitrided layer is given in table 3. Fig. 9. Imprints of microhardness measurements (the section is not etched) In accordance with the developed technological regulations, plasma precision nitriding of Avinit N and subsequent application of antifriction coating Avinit C310 full-size high-precision gear by duplex technology was carried out (). Fig. 9. Imprints of microhardness measurements (the section is not etched) When nitriding a full-size gear, a witness sample was used (Fig. 8), on which nitriding regimes were tested and which was subjected to metallurgical research [21]. 85 Mech. Adv. Technol. Vol. 5, No. 1, 2021 Table 3. The hardness of the nitrided layer No Distance from the edge, mm Hardness, Hμ, in parentheses HRC 1 0,05 603, 644 (57,5) 633 (57) 2 0,1 612 (56), 557 (53), 584 (54) 3 0,15 532, 532 (51), 516 4 0,20 524, 524 (50,7) 5 0,25 501 (49), 501, 494 (49) 6 0,30 453 (45,6) 7 0,35 466, 453 (46) 8 0,40 401 (41), 391 (39,8) 9 0,45 376, 390, Table 3. The hardness of the nitrided layer degree of accuracy 4 in accordance with the requirements of the technical documentation. 3.1. Preliminary tests The results of preliminary tests of the gear wheel with plasma nitriding and Avinit C310 coating are de- scribed in [21]. The gear was tested as part of the gearbox of the AI-450M engine on the hydraulic brake stand [21]. The total operating time during the tests was 1 hour 30 minutes, of which mode - 75 minutes, on takeoff mode – 6 minutes. The power transmitted by the engine in takeoff mode is 465 hp, while the torque on the gear is 22.67 kgf·m. The maximum specific load on the gear tooth, taking into account the end and axial overlap is 453.4 kgf/cm, while the contact stresses on the surface are 121 kgf/mm2. The microstructure of the nitrided layer is satisfac- tory (Fig. 10). Fig. 10. The microstructure of the nitrided layer (x500) After testing, the gear was dismantled to perform defecting, magnetic powder control and measurements. If any damage to the gear, including the Avinit coating, is not detected, there are weak traces of work on the coating. Antifriction coating Avinit C310 with a thickness of 1.5 μm does not distort the geometry of the tooth pro- files, in the process of the first tests in the gearbox of the AI-450M engine wear wear is not observed. Measurement of the parameters of the teeth showed a complete lack of wear. 3.2. Test of a full-size gear wheel 4501150003-02 Long-term tests of the gear wheel were carried out as part of the gearbox of the engine AI-450M № 450M-003 on the hydraulic brake stand of SE “Ivchenko-Progress” [21]. The operating time of the wheel was 40 h at a max- imum voltage of 115.06 (kgf/mm2). Fig. 10. The microstructure of the nitrided layer (x500) The measurements of the toothed ring before nitrid- ing confirm the manufacture of the gear on the 4th degree of accuracy. g ( g ) During the tests, the gear wheel 45011500003-02 was the guide for the driven wheel 45011500004-01, manufactured by standard technology (gas nitriding - grinding - copper coating) (Fig. 11). After the tests, both wheels were defective. Examination of the teeth of the wheel 45011500003-02 showed no wear of the coating on the tooth profiles, however, there was wear of the coating on the corresponding teeth of the wheel 45011500004-01. Due to the small coating thickness (2 μm), its wear did not affect the performance of the gear. Preliminary long- term tests of similar gears made by standard technology have shown that the wear of the copper coating on the teeth occurs only in the case of contact with foreign parti- cles. Thus, the combination of hard and soft coating of the teeth of the two engaged wheels showed a negative result. The reason for the wear of the copper coating is probably micro-irregularities on the surface of the teeth, which in the process of sliding surfaces when engaged cut the coating. Measurements of the toothed crown after nitriding and coating showed that changes in the geometry after plasma precision nitriding Avinit N and Avinit coating by duplex technology is not observed. Reducing the nitriding time ensures that the parameters of the gear ring gear after nitriding within the accuracy of 4 (ISO - +1328 - 1), de- signed for loaded gears of aircraft reducers. The adhesion of the coating was checked by the standard method used for galvanic coatings and was satis- factory. It is established that changes in geometry after plasma precision nitriding of Avinit N and Avinit coating by duplex technology are not observed. Avinit C310 anti- friction coating with a thickness of 1.5 μm does not dis- tort the geometry of the tooth profiles. All parameters of the gear ring, made by duplex technology Avinit, meet the 86 Mech. Adv. Technol. Vol. 5, No. 3.2. Test of a full-size gear wheel 4501150003-02 1, 2021 a b Fig. 11. Photos of gears a) gear 45011500003-02 / 4501150008-60; b) gear 45011500004-01 a Fig. 11. Photos of gears a) gear 45011500003-02 / 4501150008-60; b) gear 45011500004-01 After the tests, the gears are defective. Thus, the gear wheel 45011500003-02 with super- hard coating Avinit C310 was tested as part of the gear- box of the engine AI-450M for 40 hours. After tests of changes in the geometry of the teeth of the wheel and wear of the coating Avinit C310 was not detected, but there is wear of the copper coating of the teeth of the mat- ing gear with the appearance of a mirror luster on the sur- face of the teeth. The appearance of the teeth of the wheels showed a forgery of the coating similar to that which was on the pro- duction wheels, ie wear of the coating on only one of the wheels is not. Measurements also showed identical grind- ing of the teeth of both wheels. Conclusions 1. Plasma precision technology Avinit N allows you to perform nitriding of the finished parts without changing the size, including gears of 4 degrees of accuracy. After the production of the driven gear 4501150004-01, strengthened by duplex technology Avinit, joint tests of a pair of gears 4501150003-02 and 4501150004-01 were carried out. The tests were carried out as part of the AI-450M engine on the hydraulic brake stand of Ivchenko-Progress according to the program of equivalent cyclic tests. A pair of experimental gears was installed in the engine gearbox instead of the serial wheels of the second gearbox. The total test time of the wheels was 26 hours. 2. Nitriding time of Avinit N is 2...4 times less than with gas, with epsilon nitride phase is almost absent. 3. Coating of parts Avinit C310 increases the mi- crohardness of the surfaces of parts and reduces the coef- ficient of friction, while having sufficient adhesion to the working surfaces of the teeth of the gears and tread bearings. [5] Antifriktsionnye pokrytiya rabochikh poverkhnostei shesteren i podshipnikov. Tekhnicheskii otchet, T/o No. 4/2007-CESAR, GP “Ivchenko-Progress”, Zaporozh'e, 2007. rzamasov et al., Ionnaya khimiko-termicheskaya obrabotka splavov, Moscow, MGU im. Baumana, 1999. khnologiya proizvodstva i metody obespecheniya kachestva zubchatykh koles i peredach. SPb: Professiya, 2007. References [19] V.Popov, A.Sagalovych and V.Sagalovych, Improving the performance, reliability and service life of aviation technology products based on the innovative vacuum-plasma nanotechnologies for application of avinit functional coatings and surfaces modification: monograph. Tallinn, Scientific Route OÜ Publ., 2020. DOI: https://doi.org/10.21303/978-9916-9516-1-3 [20] O.V. Sagalovich et al., “Vacuum-plasma protective coating for turbines blades”, Mechanics and Advanced Technologies, No. 1(88), pp. 124–134 2020. http://doi.org/10.20535/2521-1943.2020.88.204675 [21] Ispytaniya pokrytiya Avinit S310 na dvigatele AI-450M. Tekhnicheskaya spravka, No. 488/2017, GP “Ivchenko-Progress”, Zaporozh'e, 2017. [22] A.V. Kononykhin et al., “Izuchenie tribologicheskikh kharakteristik mnogosloinykh Mo-S pokrytii, poluchennykh gazofaznym metodom s ispol'zovaniem metallorganicheskikh soedinenii”, Vestnik KhNADU, V. 54, pp. 44–51, 2011. [23] A.V. Sagalovich et al., “Nanesenie pokrytii na slozhnoprofil'nye pretsizionnye poverkhnosti gazofaznym metodom (CVD)”, Fizicheskaya inzheneriya poverkhnosti, T. 9, No. 3, pp. 229–236, 2011. [24] A.V. Sagalovich et al., “Izuchenie tribologicheskikh kharakteristik mnogosloinykh Mo-S pokrytii, poluchennykh gazofaznym metodom s ispol'zovaniem metallorganicheskikh soedinenii”, Tekhnologicheskie sistemy, No. 1(58), pp. 46–54, 2012. [25] A.Sagalovych, V. Sagalovych, “Mo-C multilayered CVD coatings”, Tribology in industry, V. 35, No. 4, pp. ych, V. Sagalovych, “Mo-C multilayered CVD coatings”, Tribology in industry, V. 35, No. 4, pp. 219–227, 2013. [ ] g y g y y g gy y pp [26] A. Sagalovych et al., “The Antifrictional Coatings on the Molybdenum Base”, Proc. 15th Intern. Conf. on Tribology SERBI- ATRIB ’17, Kragujevac, Serbia, 2017. [27] A. Sagalovych et al., “Development of the chemical vapor deposition process for applying molybdenum coatings on the com- ponents in assembly and engine construction”, Skhіdno-Evropeis'kii zhurnal peredovikh tekhnologіi (EEJET), No. 2/12(104), pp. 6–15, 2020. https://doi.org/10.15587/1729-4061.2020.201540 References [3] B.N. Arzamasov et al., Ionnaya khimiko-termicheskaya obrabotka splavov, Moscow, MGU im. B [4] Yu.S. Eliseev et al., Proizvodstvo zubchatykh koles gazoturbinnykh dvigatelei, Moscow: Vyssh. shkola, 2001 [5] Antifriktsionnye pokrytiya rabochikh poverkhnostei shesteren i podshipnikov. Tekhnicheskii otchet, T/o No. 4/2007-CESAR, GP “Ivchenko-Progress”, Zaporozh'e, 2007. [5] Antifriktsionnye pokrytiya rabochikh poverkhnostei shesteren i podshipnikov. Tekhnicheskii otchet, T/o No GP “Ivchenko-Progress”, Zaporozh'e, 2007. Yu.S. Eliseev, V.V. Krymov, “Chto nado dlya sozdaniya dvigatelya novogo pokoleniya”, Dvigatel', No. 3 (73), p [6] Yu.S. Eliseev, V.V. Krymov, “Chto nado dlya sozdaniya dvigatelya novogo pokoleniya”, Dvigatel', No. 3 (73), pp. 2–5, 2007. [6] Yu.S. Eliseev, V.V. Krymov, “Chto nado dlya sozdaniya dvigatelya novogo pokoleniya” [6] Yu.S. Eliseev, V.V. Krymov, “Chto nado dlya sozdaniya dvigatelya novogo pokoleniya”, Dvigatel', No. 3 (73), pp. 2–5, 2007. [7] I.F. Kravchenko et al., “Eksperimental'nye i teoreticheskie rezul'taty issledovaniya aviatsionnykh zubchatykh peredach dlya dvigatelei pyatogo i shestogo pokolenii” Aviatsionno-kosmicheskaya tekhnika i tekhnologiya No 8 (55) pp 129–134 2008 [8] O.V. Sagalovich and V.V. Sagalovich, “Sposіb іonno-plazmovogo pretsizіinogo azotuvannya poverkhon Avinit N”, No.84664, 25.10.13. [9] A.V. Sagalovich and V.V. Sagalovich, “Sposіb іonno-plazmovogo pretsizіinogo azotuvannya poverkhon' stalei ta splavіv Avinit N”, No.107408, 28.12.14. [9] A.V. Sagalovich and V.V. Sagalovich, “Sposіb іonno-plazmovogo pretsizіinogo azotuvannya poverkhon' stalei ta splavіv Avinit N”, No.107408, 28.12.14. [10] A.V. Sagalovich, V.V. Popov and V.V. Sagalovich, “Plazmove pretsizіine azotuvannya Avinit N”, Tekhnologicheskie sistemy, No. 4(89), pp. 16–26, 2019. 87 Mech. Adv. Technol. Vol. 5, No. 1, 2021 [11] Issledovanie metodov uprochneniya razrabotki NTTs “Nanotekhnologiya”, Tekhnicheskaya spravka No. 106/2015. GP “Ivchenko-Progress”, Zaporozh'e, 2015. [12] Issledovanie plazmennogo azotirovaniya shesteren. Tekhnicheskaya spravka, No. 92/2015. GP “Ivchenko-Progress”, Zapo- rozh'e, 2015. [13] A.V.Sagalovich et al., “Porіvnyal'nii analіz vtomnoї kontaktnoї mіtsnostі poverkhon', zmіtsnenikh tsementuvannyam ta іonno- plazmovim azotuvannyam Avіnіt N”, Vіsnik dvigunobuduvannya. No. 1, pp. 33–45, 2020. [14] A.V. Sagalovich et al., “Eksperimental'nye issledovaniya pokrytii tipa Avinit, Aviatsionno-kosmiche tekhnologiya. Tekhnologiya proizvodstva letatel'nykh apparatov”, T. 1, pp. 5–15, 2011. [15] A. Sagalovych et al., “Experimental research of multicomponent multilayer ion-plasma Avinit coatings”, zheneriya poverkhnosti, T. 10, No. 4, pp. 229–236, 2012. [16] A.Sagalovych et al., “Experimental research of multicomponent multilayer ion-plasma Avinit coatings”, Fizicheskaya in- zheneriya poverkhnosti, T. 11, No. 1, pp 4–17, 2013. [17] O.V. Sagalovich et al., “Znosostіike antifriktsіine pokrittya”, No. 109053, 10.07.15. [18] A.V. Sagalovich et al., “Bagatosharove, znosostіike pokrittya”, No. 108279, 10.04.15. Застосування вакуум-плазмових технологій Avinit до виготовлення повнорозмірних високоточних шестерень. Пара експеримента- льних зубчастих коліс була встановлена в редуктор двигуна замість серійних коліс другого ступеня редуктора. Сумарний час випробувань коліс склав 26 годин. Після випробувань ніяких пошкоджень шестерні, включаючи покриття Avinit, не встановлено. Антифрикційне покриття Avinit С310 товщиною 1,5 мкм не спотворює геометрію профілів зубів в процесі випробувань в складі редуктора двигуна АІ-450М. Обмір параметрів зубів показав повну відсутність зносу. Випробування виготовлених по дуплексній технології Avinit шестерень здійснювалося в складі редуктора двигуна АІ-450М на гідротормозному стенді ДП “Івченко-Прогрес” за програмою еквівалентно-циклічних випробувань. Пара експеримента- льних зубчастих коліс була встановлена в редуктор двигуна замість серійних коліс другого ступеня редуктора. Сумарний час випробувань коліс склав 26 годин. Після випробувань ніяких пошкоджень шестерні, включаючи покриття Avinit, не встановлено. Антифрикційне покриття Avinit С310 товщиною 1,5 мкм не спотворює геометрію профілів зубів в процесі випробувань в складі редуктора двигуна АІ-450М. Обмір параметрів зубів показав повну відсутність зносу. Ключові слова: розробка дуплексних технологій Avinit, властивості азотувального шару, характеристики покриттів, сте- ндові випробування виготовлених по дуплексній технології Avinit високоточних шестерень. Применение вакуум-плазменных технологий Avinit к изготовлению полноразмерных высокоточных шестерен. А.В. Сагалович, В.В. Попов, В.В. Сагалович, С.Ф. Дудник, А.Б. Единович. Аннотация. Разработаны дуплексные технологии Avinit, объединяющие плазменное азотирование Avinit N окончательно готовых высокоточных деталей с последующим нанесением сверхтвердых антифрикционных покрытий Avinit в едином технологическом процессе Благодаря отсутствию на азотируемой поверхности хрупкого слоя после прецизионного азотирования, сохранению исход- ных геометрических размеров, не требующих дальнейшей механической доработки, и совместимости процессов плазмен- ного прецизионного азотирования Avinit N и вакуум-плазменного нанесения функциональных покрытий Avinit С дуплексные технологии позволяют обеспечить нанесение прочносцепленных, высококачественных покрытий. Исследовано влияние дуплексного процесса на размеры деталей при плазменном азотировании высокоточных шестерен и нанесении функциональных покрытий Avinit С, изучены свойства азотированного слоя и параметры покрытий Avinit. Плазменная прецизионная технология Avinit N позволяет выполнять азотирования окончательно готовых деталей без из- менения размеров, в том числе шестерен по 4 степени точности. Время азотирования Avinit N в 2 – 4 раза меньше, чем при газовом азотировании. Покрытие деталей Avinit С310 повышает микротвердость поверхностей деталей и снижает коэффи- циент трения, при этом обладает достаточной адгезией к рабочим поверхностям зубьев шестерен и беговых дорожек подшип- ников. Проведено изготовление высокоточных зубчатых колес по степени точности 4 с использованием дуплексных технологий Avinit. Анализ результатов показывает, что в пределах точности измерения никаких изменений профилей зубьев, их распо- ложение на зубчатом венце, а также расположение зубчатого венца относительно измерительных баз не наблю-дается. Плазменное азотирование позволяет сократить время азотирования более чем в два раза по сравнению с газовым азоти- рованием, при этом толщина слоя хрупкой фазы с максимальной поверхностной твердостью обеспечивается в пределах заданных величин для обеспечения необходимых показателей контактной и изгибной длительной прочности при изготов- лении зубчатых колес по степени точности 4 без шлифовки после азотирования. Измерения зубчатого венца после азотирования и покрытия показали, что изменений геометрии шестерни, обработанной по дуплексной технологии, не наблюдается. Антифрикционное покрытие Avinit С310 толщиной 1,5 мкм не искажает гео- метрию профилей зубьев. Все параметры зубчатого венца, изготовленного по дуплексной технологии Avinit, соответ- ствуют степени точности 4 соответствии с требованиями технической документации. Испытания изготовленных по дуплексной технологии Avinit шестерен осуществлялось в составе редуктора двигателя АИ- 450М на гидротормозном стенде ГП “Ивченко-Прогресс” по программе эквивалентно-циклических испытаний. Пара экс- периментальных зубчатых колес была установлена в редуктор двигателя вместо серийных колес второй ступени редук- тора. Суммарное время испытаний колес составило 26 часов. После испытаний никаких повреждений шестерни, включая покрытие Avinit, не установлено. Антифрикционное покрытие Avinit С310 толщиной 1,5 мкм не искажает геометрию профилей зубьев в процессе испытаний в составе редуктора двигателя АИ-450М. Обмер параметров зубов показал полное отсутствие износа. Застосування вакуум-плазмових технологій Avinit до виготовлення повнорозмірних високоточних шестерень. О. В. Сагалович, В. В. Попов, В. В. Сагалович, С. Ф. Дудник, А. Б. Єдинович Анотація. Розроблено дуплексні технології Avinit, які об'єднують плазмове азотування Avinit N остаточно готових висо- коточних деталей з подальшим нанесенням надтвердих антифрикційних покриттів Avinit в єдиному технологічному проце- сі Завдяки відсутності на азотуючій поверхні крихкого шару після прецизійного азотування, збереженню вихідних геометри- чних розмірів, які не потребують подальшої механічного доопрацювання, і сумісності процесів плазмового високоточного азотування Avinit N і вакуум-плазмового нанесення функціональних покриттів Avinit З дуплексні технології дозволяють забезпечити нанесення міцно-щеплених, високоякісних покриттів. Досліджено вплив дуплексного процесу на розміри дета- лей при плазмовому азотуванні високоточних шестерень і нанесенні функціональних покриттів Avinit С, вивчені власти- вості азотувального шару і параметри покриттів Avinit. Плазмова прецизійна технологія Avinit N дозволяє виконувати азотування остаточно готових деталей без зміни розмірів, в тому числі шестерень по 4 ступеню точності. Час азотування Avinit N в 2...4 рази менше, ніж при газовому азотуванні. Покриття деталей Avinit С310 підвищує мікротвердість поверхонь деталей і знижує коефіцієнт тертя, при цьому володіє достатньою адгезією до робочих поверхонь зубів шестерень і бігових доріжок підшипників. Проведено виготовлення високоточних зубчастих коліс за ступенем точності 4 з використанням дуплексних технологій Avinit. Аналіз результатів показує, що в межах точності вимірювання ніяких змін профілів зубів, їх розташування на зубчастому вінці, а також розташування зубчастого вінця щодо вимірювальних баз не спостерігається. Плазмове азотування дозволяє скоротити час азотування більш ніж в два рази в порівнянні з газовим азотуванням, при цьому товщина шару тендітної фази з максимальною поверхневою твердістю забезпечується в межах заданих величин для забезпечення необхідних показників контактної і згинальної тривалої міцності при виготовленні зубчастих коліс за ступенем точності 4 без шліфування після азотування.Вимірювання зубчастого вінця після азотування і покриття показа- 88 Mech. Adv. Technol. Vol. 5, No. 1, 2021 ли, що змін геометрії шестерні, обробленої по дуплексной технології, не спостерігається. Антифрикційне покриття Avinit С310 товщиною 1,5 мкм не спотворює геометрію профілів зубів. Всі параметри зубчастого вінця, виготовленого за дупле- ксною технологією Avinit, відповідають ступеню точності 4 відповідно до вимог технічної документації. ли, що змін геометрії шестерні, обробленої по дуплексной технології, не спостерігається. Антифрикційне покриття Avinit С310 товщиною 1,5 мкм не спотворює геометрію профілів зубів. Всі параметри зубчастого вінця, виготовленого за дупле- ксною технологією Avinit, відповідають ступеню точності 4 відповідно до вимог технічної документації. Випробування виготовлених по дуплексній технології Avinit шестерень здійснювалося в складі редуктора двигуна АІ-450М на гідротормозному стенді ДП “Івченко-Прогрес” за програмою еквівалентно-циклічних випробувань. Применение вакуум-плазменных технологий Avinit к изготовлению полноразмерных высокоточных шестерен. Ключевые слова: разработка дуплексных технологий Avinit, свойства азотированного слоя, характерис стендовые испытания изготовленных по дуплексной технологии Avinit высокоточных шестерен.
https://openalex.org/W2611252229	https://europepmc.org/articles/pmc5419906?pdf=render	English	null	Increased risk of ventricular tachycardia and cardiovascular death in patients with myocarditis during the long-term follow-up	Medicine	2,017	cc-by	5,234	Abstract The incidence of acute myocarditis complicated with ventricular tachycardia (VT) is unknown. This study aimed to investigate the association between myocarditis and the incidence of VT and mortality. We also aimed to determine the independent predictors that increased the VT risk in those patients. From 2000 to 2004, 13,250 patients with a history of myocarditis were identiﬁed from the Taiwan National Health Insurance Research Database. The same number of individuals without heart disease with a matched sex and underlying diseases were selected as the control group. The long-term risks of life-threatening ventricular arrhythmias and mortality in patients with a history of myocarditis were investigated by an adjusted Cox proportional hazards regression. After a mean follow-up of 10.4±2.94 years (interquartile range: 12, 10.19–12), the myocarditis patients showed a higher incidence of new onset VT events compared with healthy controls (5.4% [519 per 100,000 person-year] in the myocarditis group vs, 0.47% [43 per 100,000 person- year] in the healthy controls; adjusted hazard ratio [HR]: 16.1, 95% conﬁdence interval [CI]: 12.4–20.9; P<.001). A higher incidence of cardiovascular death was noted in the myocarditis group than healthy controls (6.52% vs 3.18%; HR: 2.42, 95% CI: 2.14–2.73; P<.001) after adjusting for the multivariate confounders including sex, age, underlying comorbidities, and medications. The results of this study suggested that there was higher incidence of life-threatening VT and mortality during the very long-term follow-up in patients with a history of myocarditis. Future work should focus on an in-depth risk stratiﬁcation of VT in myocarditis patients. Abbreviations: ACEi = angiotensin-converting enzyme inhibitor, ARB = angiotensin receptor II blocker, BB = beta-blocker, CI = conﬁdence interval, CKD = chronic kidney disease, COPD = chronic obstructive pulmonary disease, DM = diabetes mellitus, HR = hazard ratio, HTN = hypertension, ICD = implantable cardioverter deﬁbrillator, ICD9-CM = International Classiﬁcation of Diseases, 9th Revision—Clinical Modiﬁcation, NHIRD = National Health Insurance Research Database, VT = ventricular tachycardia. Keywords: mortality, myocarditis, ventricular tachycardia Keywords: mortality, myocarditis, ventricular tachycardia Increased risk of ventricular tachycardia and cardiovascular death in patients with myocarditis during the long-term follow-up A national representative cohort from the National Health Insurance Research Database Abigail Louise D. Te, MDa, Tao-Cheng Wu, MDa,b, Yenn-Jiang Lin, MD, PhDa,b,∗, Yun-Yu Chen, MPHa,c, Fa-Po Chung, MDa,b, Shih-Lin Chang, MD, PhDa,b, Li-Wei Lo, MDa,b, Yu-Feng Hu, MDa,b, Ta-Chuan Tuan, MDa,b, Tze-Fan Chao, MDa,b, Jo-Nan Liao, MDa,b, Kuo-Liong Chien, MD, PhDc, Chin-Yu Lin, MDa, Yao-Ting Chang, MDa, Shih-Ann Chen, MDa,b Abigail Louise D. Te, MDa, Tao-Cheng Wu, MDa,b, Yenn-Jiang Lin, MD, PhDa,b,∗, Yun-Yu Chen, MPHa,c, Fa-Po Chung, MDa,b, Shih-Lin Chang, MD, PhDa,b, Li-Wei Lo, MDa,b, Yu-Feng Hu, MDa,b, Ta-Chuan Tuan, MDa,b, Tze-Fan Chao, MDa,b, Jo-Nan Liao, MDa,b, Kuo-Liong Chien, MD, PhDc, Chin-Yu Lin, MDa, Yao-Ting Chang, MDa, Shih-Ann Chen, MDa,b 1. Introduction ALDT and T-CW have contributed equally to the article. ALDT and T-CW have contributed equally to the article. Myocarditis is a common cardiac disease that is identiﬁed in up to 9% of routine postmortem examinations.[1] It can present with heterogeneous clinical manifestations from nonspeciﬁc symp- toms to a rapidly deteriorating cardiac function and life- threatening arrhythmias.[2] In patients with a benign course of the disease, the cardiac function may completely recover or progress to chronic dilated cardiomyopathy.[3] Long-term follow-up studies[4–6] of patients with a history of acute myocarditis often have a 5- or 6-year follow-up period, and those studies looked into the association of the development of chronic dilated cardiomyopathy and the risk of sudden cardiac death. Other long-term studies were limited to a certain etiology of the myocarditis and identiﬁcation of the long-term predictors of the mortality using several parameters from diagnostic studies.[4,7] However, the incidence of new-onset ventricular tachycardia (VT) during a 10-year follow-up period especially in patients who recovered from acute myocarditis without any evident clinical cardiovascular (CV) sequelae is presently unknown. In addition, no study has been able to show the overall mortality risk in these patients during the very long-term follow-up. The purpose of this study was to investigate the Funding/support: The work was supported by the Research Foundation of Cardiovascular Medicine (RFCM 96-02-018, 97-02-029, 98-01-020, 98-02-032, 99-01-022, 100-01-004, 100-02-011, 100-02-022, and 101-01-001) and Taipei Veterans General Hospital (V102C-128, V103E7-003, VN103-04, V103C-042, and V104B-018). Funding/support: The work was supported by the Research Foundation of Cardiovascular Medicine (RFCM 96-02-018, 97-02-029, 98-01-020, 98-02-032, 99-01-022, 100-01-004, 100-02-011, 100-02-022, and 101-01-001) and Taipei Veterans General Hospital (V102C-128, V103E7-003, VN103-04, V103C-042, and V104B-018). The authors have no conﬂicts of interest to disclose. a Division of Cardiology, Department of Medicine, Taipei Veterans General Hospital, b School of Medicine, Institute of Clinical Medicine and Cardiovascular Research Center, National Yang-Ming University, c Institute of Epidemiology and Preventive Medicine College of Public Health, National Taiwan University, Taipei, Taiwan. ∗Correspondence: Yenn-Jiang Lin, Division of Cardiology, Department of Medicine, Taipei Veterans General Hospital, No. 201, Sec. 2, Shih-Pai Road, Taipei, Taiwan (e-mail: linyennjiang@gmail.com). Copyright © 2017 the Author(s). Published by Wolters Kluwer Health, Inc. This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Medicine ® Medicine ® Medicine ® OPEN Observational Study http://dx.doi.org/10.1097/MD.0000000000006633 3.2. Incidence of events The patients with myocarditis had a higher incidence of new onset VT events than the healthy controls (5.40% [519 per 100,000 person-year] in myocarditis group vs 0.47% [43 per 100,000 person-year] in healthy controls; adjusted HR: 16.06, 95% CI: 12.37–20.86; P<.001). Higher incidences of CV death (6.52% vs 3.18%; adjusted HR: 2.42, 95% CI: 2.14–2.73; P<.001; Table 2) and all-cause mortality (24.5% vs 18.9%; adjusted HR: 1.41, 95% CI: 1.33–1.49; P<.001) were also noted in the myocarditis group than the healthy cohort. The incidence of ICD implantations was also signiﬁcantly higher in the 2.4. Statistical analysis The normally distributed continuous variables were presented as the mean values and standard deviation and were compared using a Student t test. The non-normally distributed variables were compared using the Mann–Whitney U test. Frequencies were compared using the chi-square test. The incidence rates of CV events were calculated as the number of cases per 100,000 person-years of follow-up. In order to minimize the impact of the confounding factors on the clinical characteristics, we employed the propensity analysis and matching technique. We matched the pairs one-to-one with identical propensity scores with a 0.01 caliper width for the sex, HTN, DM, CKD, COPD, hyperlipid- emia, and thyroid disease. 2.1. Study design and participants This study was a population-based retrospective cohort study. From January 1, 2000 to December 31, 2004, a total of 13,250 patients aged 18 years and older, who were diagnosed with myocarditis, were identiﬁed from the NHIRD according to the International Classiﬁcation of Diseases, 9th Revision—Clinical Modiﬁcation (ICD9-CM) codes (422). The diagnosis of myocarditis must be recorded twice in the outpatient records or at least once in the inpatient records. On the same index date, the same number of health controls without prior structural heart disease, matched by the sex, history of hypertension (HTN), diabetes mellitus (DM), chronic obstructive pulmonary disease (COPD), chronic kidney disease (CKD), hyperlipidemia, and thyroid disease, were selected to be the control group for each study patient. Patients who were diagnosed with ischemic heart disease, heart failure, valvular heart disease, congenital heart disease, overt cardiogenic shock requiring vasopressors at the initial presentation had a history of ventricular arrhythmia or previous implantable cardioverter deﬁbrillator (ICD) implanta- tion, and those with systemic inﬂammatory diseases known to be associated with myocardial involvement were excluded from the study. The comorbid conditions of each individual were retrieved from the medical claims database based on the ICD9-CM codes. The event-free survival curve was plotted using the Kaplan– Meier method with the statistical signiﬁcance examined by the log-rank test. The Cox proportional-hazards regression was used to compare the hazard ratios (HR) with 95% conﬁdence intervals (CIs) for the outcomes. Potential confounders were adjusted via 3 models. Model 1: age and sex; Model 2: Model 1 plus HTN, DM, COPD, CKD, hyperlipidemia, and thyroid disease; Model 3: Model 2 plus medications, including angiotensin-converting enzyme inhibitors (ACEis), angiotensin receptor II blockers (ARBs), and beta-blockers (BBs). The level of statistical signiﬁcance was set at a 2-tailed alpha level <0.05. The analyses were performed with SAS version 9.3 software (SAS Institute, Cary, NC). 3.1. Patient characteristics This study used the Taiwan NHIRD to determine the risk of ventricular tachycardia (VT) and CV death in patients with myocarditis during the long-term follow-up (from 2000 to 2011). The Taiwan Collaboration Center of Health Information Application, Ministry of Health and Welfare, provided the entire datasets used in this study. The Taiwan’s National Health Insurance (NHI) program enrolled 23 million people, which covered 99% of the country’s population and contained data on utilization of all NHI resources, including outpatient visits, hospital care, prescribed medications, and the National Death Registry. It contained all the medical claims data for 1000,000 beneﬁciaries, who were randomly sampled from the 25.68 million enrollees under the NHI program. These random samples have been conﬁrmed by the NHRI to be representative of the Taiwanese population. The protocol was reviewed and approved by the Research Ethics Committee of National Taiwan University Hospital (NTUH-Rec Number: 201305044W [Institutional Review Board reference]). In addition, we obtained permission for the rights from the National Research Institute for the Department of Health and the Health Promotion Administration, Ministry of Health and Welfare. A total of 13,250 patients with a history of myocarditis and 13,250 healthy controls without a prior history of structural heart disease were identiﬁed as the study population with a mean follow-up of 10.4±2.94 years (interquartile range: 12, 10.19–12). The characteristics of the study population are shown in Table 1. The baseline characteristics of the two populations did not signiﬁcantly differ except for the age wherein the patients with myocarditis were younger than the healthy cohort (myocarditis group, 43±27 years old vs healthy cohort, 44±26 years old, P<.01). In addition, the use of antihyperten- sive medications, particularly ACEis, ARBs, and BBs, were signiﬁcantly more frequent in the myocarditis group than in the healthy cohorts (ACEi: 15.11% patients with myocarditis vs 0.01% healthy cohorts, P<.001; ARB: 3.97% vs 0.02%, P<.001; BB: 16.96% vs 0.04%, P<.001). 1. Introduction Received: 1 December 2016 / Received in ﬁnal form: 21 March 2017 / Accepted: 25 March 2017 1 1 Te et al. Medicine (2017) 96:18 Medicine National Death Registry. The time to implantation of an ICD was investigated as a secondary endpoint. National Death Registry. The time to implantation of an ICD was investigated as a secondary endpoint. association between acute myocarditis and the incidence of VT and mortality during the long-term follow-up period from the National Health Insurance Research Database (NHIRD) of Taiwan. association between acute myocarditis and the incidence of VT and mortality during the long-term follow-up period from the National Health Insurance Research Database (NHIRD) of Taiwan. 3.3. Event-free survival The Kaplan–Meier event-free survival curves for new-onset VT events, CV death, all-cause mortality, and ICD implanta- tions comparing patients with and without myocarditis are shown in Fig. 1A–D. There were signiﬁcant differences in the new-onset VT events, CV death, and all-cause mortality between the two groups (Fig. 1A–C) with the myocarditis groups showing increased new-onset VT events and decreased survival compared to the healthy controls. In addition, the overall risk for mortality (Fig. 1C) for the two groups was 2.3. Study endpoints during the follow-up The follow-up period ended when the subjects developed new onset VT, death, or lived beyond December 31, 2011. The primary endpoints were the time to the development of new-onset VT and the time to CV death/all-cause mortality during the follow-up. Death was conﬁrmed by referencing the Taiwan’s 2 Te et al. Medicine (2017) 96:18 www.md-journal.com Table 1 Characteristics of patients with and without myocarditis. Variables Without myocarditis (n=13,250) With myocarditis (n=13,250) P ∗ Age, y, mean±SD 44±26 43±27 .01 Gender (male) 6305 (47.60) 6309 (47.06) .96 Underlying disease HTN 2440 (18.40) 2410 (18.20) .63 DM 972 (7.34) 972 (7.34) >.99 COPD 1156 (8.72) 1171 (8.84) .74 CKD 85 (0.64) 75 (0.57) .74 Hyperlipidemia 618 (4.66) 635 (4.79) .62 Thyroid disease 243 (1.83) 243 (1.83) >.99 ACEi 1 (0.01) 2002 (15.11) <.001 ARB 2 (0.02) 526 (3.97) <.001 Beta-blocker 5 (0.04) 2247 (16.96) <.001 Values are in n, %. ACEi = angiotensin-converting enzyme inhibitor, ARB = angiotensin II receptor blocker, BB = beta-blocker, CI = conﬁdence interval, CKD = chronic kidney disease, COPD = chronic obstructive pulmonary disease, DM = diabetes mellitus, HTN indicates = hypertension. ∗P values <.05 were considered to indicate statistical signiﬁcance. Values are in n, %. ACEi = angiotensin-converting enzyme inhibitor, ARB = angiotensin II receptor blocker, BB = beta-blocker, CI = conﬁdence interval, CKD = chronic kidney disease, COPD = chronic obstructive pulmonary disease, DM = diabetes mellitus, HTN indicates = hypertension. ∗P values <.05 were considered to indicate statistical signiﬁcance. myocarditis group (0.13% vs 0.02%; adjusted HR: 12.07, 95% CI: 2.74–53.08, P<.001) than the healthy control population after adjusting for multivariate confounders including the sex, age, underlying comorbidities, and medications (Table 2). initially similar (from index event up to follow-up of 5 years). However, 5 years onward in the course of the follow-up, the Kaplan–Meier curve demonstrated a divergence in the survival curve between the myocarditis group and healthy controls, wherein the myocarditis group had a signiﬁcantly increased overall mortality risk (log rank P<.001), whereas the healthy controls’ mortality risk remained the same. The risk for the ICD implantations was also signiﬁcantly higher in the myocarditis group than in the healthy controls during long-term follow-up (log rank P<.001). Model 0: crude effect size; Model 1: age and sex. Model 2: Model 1+underlying comorbidities: hypertension, diabetes mellitus, chronic obstructive pulmonary disease, chronic kidney disease, hyperlipidemia, and thyroid disease. Model 3: Model 2+medications: ACEi, ARB, and BB. ACEi = angiotensin-converting enzyme inhibitor, ARB = angiotensin II receptor blocker, BB = beta-blocker, CV = cardiovascular, ICD = implantable cardiac deﬁbrillator, VT = ventricular tachycardia. ∗P values <.05 were considered to indicate statistical signiﬁcance. 3.4. Predictors of the occurrence of ventricular arrhythmias Those who developed VT were mostly males (with VT, 55.30% vs without VT, 47.20%, P<.001), had DM (10.20% vs 7.17%, P=.01), CKD (2.51% vs 0.45%, P<.001), hyperlipid- emia (14.30% vs 4.25%, P<.001), and a signiﬁcantly lesser use of ACEis (4.05% vs 15.7%, P<.001), ARBs (2.23% vs 4.07%, P<.001), and BBs (4.89% vs 17.7%, P<.001). A multivariable Cox proportional hazard regression analysis revealed that the independent predictors that were signiﬁcantly associated with new-onset VT events in patients with a history of myocarditis had an older age (adjusted HR: 1.003, 95% CI: 1.00–1.006, P=.04), male gender (adjusted HR: 1.33, 95% CI: 1.15–1.54, P<.001), presence of DM (adjusted HR: 1.40, 95% CI: 1.09–1.79, P=.01), CKD (adjusted HR: 2.91, 95% CI: 1.81–4.68, P<.001), hyperlipidemia (adjusted HR: 3.05, 95% CI: 2.46–3.78, Table 3. Those who developed VT were mostly males (with VT, 55.30% vs without VT, 47.20%, P<.001), had DM (10.20% vs 7.17%, P=.01), CKD (2.51% vs 0.45%, P<.001), hyperlipid- emia (14.30% vs 4.25%, P<.001), and a signiﬁcantly lesser use of ACEis (4.05% vs 15.7%, P<.001), ARBs (2.23% vs 4.07%, P<.001), and BBs (4.89% vs 17.7%, P<.001). A multivariable Cox proportional hazard regression analysis revealed that the independent predictors that were signiﬁcantly associated with new-onset VT events in patients with a history of myocarditis had an older age (adjusted HR: 1.003, 95% CI: 1.00–1.006, P=.04), male gender (adjusted HR: 1.33, 95% CI: 1.15–1.54, P<.001), presence of DM (adjusted HR: 1.40, 95% CI: 1.09–1.79, P=.01), CKD (adjusted HR: 2.91, 95% CI: 1.81–4.68, P<.001), hyperlipidemia (adjusted HR: 3.05, 95% CI: 2.46–3.78, 3.4. Predictors of the occurrence of ventricular arrhythmias The characteristics of the patients with myocarditis who developed new-onset VT during the follow-up are shown in Table 2 Incidence rate of primary and secondary outcomes in the patient population. Outcomes Variables Total numbers Event, % Incidence rate (per 100,000 PY) Models Hazard ratio (95% CI) P ∗ VT No myocarditis 13,250 62 (0.47) 43 – 1 (Reference) – With myocarditis 13,250 716 (5.41) 519 0 12.1 (9.30–15.6) <.001 1 12.1 (9.35–15.7) <.001 2 12.1 (9.35–15.7) <.001 3 16.1 (12.37–20.9) <.001 CV death No myocarditis 13,250 421 (3.18) 293 – 1 (Reference) – With myocarditis 13,250 864 (6.52) 613 0 2.12 (1.88–2.38) <.001 1 2.10 (1.87–2.36) <.001 2 2.10 (1.87–2.36) <.001 3 2.42 (2.14–2.73) <.001 All-cause mortality No myocarditis 13,250 2497 (18.90) 1737 – 1 (Reference) – With myocarditis 13,250 3243 (24.50) 2301 0 1.33 (1.26–1.40) <.001 1 1.30 (1.24–1.37) <.001 2 1.30 (1.24–1.37) <.001 3 1.41 (1.33–1.49) <.001 ICD implantation No myocarditis 13,250 2 (0.02) 1 – 1 (Reference) – With myocarditis 13,250 17 (0.13) 12 0 2.98 (0.93–9.50) .07 1 8.68 (2.00–37. 6) .01 2 8.64 (2.00–37.4) .01 3 12.1 (2.74–53.1) <.001 Model 0: crude effect size; Model 1: age and sex. Model 2: Model 1+underlying comorbidities: hypertension, diabetes mellitus, chronic obstructive pulmonary disease, chronic kidney disease, hyperlipidemia, and thyroid disease. Model 3: Model 2+medications: ACEi, ARB, and BB. ACEi = angiotensin-converting enzyme inhibitor, ARB = angiotensin II receptor blocker, BB = beta-blocker, CV = cardiovascular, ICD = implantable cardiac deﬁbrillator, VT = ventricular tachycardia. ∗P values <.05 were considered to indicate statistical signiﬁcance. 3 3 Te et al. Medicine (2017) 96:18 Medicine Figure 1. Kaplan–Meier event-free survival curves for (A) new-onset ventricular tachycardia events, (B) cardiovascular death, (C) all-cause mortality, and (D implantable cardioverter deﬁbrillator implantations comparing the patients with and without myocarditis. In the log-rank test, a P value of <.05 was considered as signiﬁcant. Figure 1. Kaplan–Meier event-free survival curves for (A) new-onset ventricular tachycardia events, (B) cardiovascular death, (C) all-cause mortality, and (D) implantable cardioverter deﬁbrillator implantations comparing the patients with and without myocarditis. In the log-rank test, a P value of <.05 was considered as signiﬁcant P<.001), and lesser use of ACEis (adjusted HR: 0.25, 95% CI: 0.17–0.37, P<.001), ARBs (adjusted HR: 0.53, 95% CI: 0.32–0.88, P=.01), and BBs (adjusted HR: 0.26, 95% CI: 0.19–0.37, P<.001). Table 3. 4.1. Main ﬁndings this study showed that these patients have a 3-fold increased risk of ICD implantations with a signiﬁcantly greater increased mortality than in the healthy cohort. The patients who may have required primary prevention for ICD implantations may show a higher CV mortality. Based on the Taiwan NHI, ICD implantations are indicated only for secondary prevention and not for primary prevention of VT events. This may be one of the reasons to explain the high mortality rate in this study. This study showed that the VT may occur 5 to 10 years after the index event of myocarditis, and the secondary prevention strategy may put these patients at risk for VT events. 4.1. Main ﬁndings The main ﬁndings of the study are as follows. First, from the national database, this study suggested a higher incidence of life- threatening VT, mortality, and ICD implantations during the very long-term follow-up in patients with a history of acute myocarditis, after adjusting for the multivariate confounders. Second, the predictors of new-onset of VT were a younger age, 4 Te et al. Medicine (2017) 96:18 www.md-journal.com Table 3 Characteristics of patients with myocarditis with and without ventricular tachycardia. Table 3 Characteristics of patients with myocarditis with and without ventricular tachycardia. Table 3 Characteristics of patients with myocarditis with and without ventricular tachycardia. Multivariate Analysis Variables Without VT (n=12,534) With VT (n=716) P ∗ Adjusted HR (95% CI) P ∗ Age, y, x±SD 43±27 42±22 <.001 1.00 (1.00–1.01) .04 Gender (male) 5913 (47.20) 396 (55.30) <.001 1.33 (1.15–1.54) <.001 Underlying diseases HTN 2308 (18.40) 102 (14.3) .001 1.04 (0.83–1.30) .75 DM 899 (7.17) 73 (10.20) .001 1.40 (1.09–1.79) .01 COPD 1107 (8.83) 64 (8.94) .92 1.07 (0.83–1.39) .60 CKD 57 (0.45) 18 (2.51) <.001 2.91 (1.81–4.68) <.001 Hyperlipidemia 533 (4.25) 102 (14.30) <.001 3.05 (2.46–3.78) <.001 Thyroid disease 225 (1.80) 18 (2.51) .16 1.25 (0.78–1.99) .36 ACEI 1973 (15.7) 29 (4.05) <.001 0.25 (0.17–0.37) <.001 ARB 510 (4.07) 16 (2.23) .01 0.53 (0.32–0.88) .01 Beta blocker 2212 (17.7) 35 (4.89) <0.001 0.26 (0.19–0.37) <.001 Values are in n, %. ACEI = angiotensin-converting enzyme inhibitor, ARB = angiotensin II receptor blocker, BB = beta-blocker, CI = conﬁdence interval, CKD = chronic kidney disease, COPD = chronic obstructive pulmonary disease, DM = diabetes mellitus, HTN = hypertension, VT = ventricular tachycardia. ∗P values <.05 were considered to indicate statistical signiﬁcance. Table 3 Characteristics of patients with myocarditis with and without ventricular tachycardia. Values are in n, %. ACEI = angiotensin-converting enzyme inhibitor, ARB = angiotensin II receptor blocker, BB = beta-blocker, CI = conﬁdence interval, CKD = chronic kidney disease, COPD = chronic obstructive pulmonary disease, DM = diabetes mellitus, HTN = hypertension, VT = ventricular tachycardia. ∗P values <.05 were considered to indicate statistical signiﬁcance. male gender, presence of DM, CKD, hyperlipidemia, and a lesser use of ACEis, ARBs, and BBs. Third, the concomitant use of ACEis, ARBs, and BBs may provide a protective role from life- threatening VT during the long-term follow-up in patients with a previous history of myocarditis. 4.2. Compared with the previous ﬁndings Myocarditis contributes to the global burden of CV disease primarily through sudden cardiac death and dilated cardiomy- opathy. Accurate population-based estimates of the myocarditis incidence and prevalence are not directly available in any world region.[8] The long-term follow-up studies in patients with acute myocarditis with preserved left ventricle systolic function mostly focused on identifying the predictors of the development of dilated cardiomyopathy,[5] which is the most common long-term sequela.[9] 4.3. Potential mechanism of VTs Although VT is an uncommon initial manifestation of myocar- ditis, it often develops during the long-term follow-up, but few studies have reported this.[10] The mechanism of VT may be attributed to the chronic myocardial inﬂammatory process secondary to pathogenic autoimmunity that may continue even after myocardial recovery.[11,12] The mechanism of arrhythmo- genesis seems to be the formation of micro-reentry circuits, favored by myocyte injury and replacement ﬁbrosis[13] and triggered activity mainly due to the proarrhythmic effects of cytokines which may continue even in the absence of overt inﬂammation.[12] The environment surrounding the myocytes could also inﬂuence the electrophysiological properties of the myocardium, leading to the phenomenon known as electrical remodeling.[13] These changes may completely or partially reverse after the healing of the inﬂammatory process. However, the exact duration and extent of this process are unknown inasmuch as the natural history of VTs in myocarditis has not been adequately studied and documented. In this study, we found that patients with a history of acute myocarditis had a 12-fold increase in new-onset VT during the very long-term follow-up compared to the healthy population without structural heart disease. This study also showed that those with a history of myocarditis had a 2-fold and 1.33-fold increase in the CV mortality and all-cause mortality, respectively. The overall survival for both groups was initially similar as depicted in the Kaplan–Meier curve (Fig. 1C). However, during the course of the follow-up, particularly 5 years onward, the survival curve started to progressively decline in the myocarditis group with a signiﬁcantly increased risk of overall mortality (25%) compared to the healthy cohort (19%). Previous studies have reported that acute myocarditis, though patients may initially present less ill,[4] causes substantial mortality with a 21% 1-year mortality rate after the presentation of myocarditis.[5,9] These mortalities were due to chronic dilated cardiomyopathy secondary to a chronic myocardial inﬂammatory process. So far, no study has been able to show the long-term overall mortality risk for patients who have recovered from acute myocarditis without any evident clinical CV sequelae. Our study showed that those groups of patients have a signiﬁcantly decreased long-term survival. Whether or not occult persistent inﬂammation plays a role in contributing to the overall long-term mortality in those patients, this distinction cannot be made based on the clinical grounds alone; thus, further study is recommended. In addition, Medicine Medicine were several limitations to our study. First, the diagnosis of myocarditis was based on the ICD-9 codes and was not further conﬁrmed with endomyocardial biopsy results. Therefore, the diagnostic accuracy of myocarditis cannot be fully established. Second, our data did not include all the various therapies that patients may have received over the 10-year follow-up period. We could only account for the use of ACEis, ARBs, and BBs in our study population. Lastly, although we adjusted for confounders using the Cox proportional-hazard regression analysis, the retrospective nature of this study limits some of the conclusions that we can derive from the data. Future studies are recommended to show the causality of the independent predictors for new-onset VT during the long-term follow-up in patients with a history of acute myocarditis. it is likely that men have a greater propensity to ventricular arrhythmias than women.[14] Numerous studies in the general population have pointed out that men experience a higher rate of ventriculararrhythmiasandsuddendeathcomparedtowomen.[14] The differences in the electrophysiologic properties related to sex hormones may, at least in part, explain the gender-speciﬁcity propensity to ventricular arrhythmias.[15] p p y y DM,[16] CKD,[17] and hyperlipidemia[18] may individually or altogether overlap to increase the myocardial vulnerability to ventricular arrhythmias. Increases in the proinﬂammatory cytokines, inﬂammatory mediators, and reactive oxygen species during the disease process have direct effects on the myocardium resulting in myocardial ﬁbrosis. This provides an additional substrate for an increased electrical instability and disturbances in the repolarization.[19] Contrary to our knowledge that HTN is a risk factor for sudden cardiac death caused by ventricular tachycardia and ﬁbrillation, our study showed that the presence of HTN in these group of patients have no signiﬁcant predictive impact on the long-term risk for VT events. However, the intake of antihypertensive medications prescribed for HTN in these patients, particularly, ACEis, ARBs, and BBs may have provided protection from a long-term risk for VT events. Studies have shown that the remodeling of the ventricle that often occurs both in acute (i.e., myocarditis) and chronic (i.e., HTN and cardiomyopathy) clinical conditions[19] may continue for months and years after the initial insult, regardless of the etiology. This remodeling can result in an increase in the susceptibility of developing ventricular arrhythmias. 4.5. Clinical Implications [7] Marholdt H, Wagner A, Deluigi CC, et al. Presentation, patterns of myocardial damage, and clinical course of viral myocarditis. Circulation 2006;114:1581–90. The natural history of myocarditis is as varied as its clinical presentation. The results of this study may have some important clinical implications. Our ﬁndings suggest that several years after admission for myocarditis, increasing events of new-onset VT and mortality could be observed in these patients. This implies that closely monitored outpatient follow-up visits may be necessary in order to provide timely management strategies to prevent the development or control of DM, CKD, and hyperlipidemia, especially among younger males. Furthermore, this study also showed the protective role of ACEis, ARBs, and BBs in preventing future VT events in those patients. Thus, for patient who has a history of myocarditis, these medications may be prescribed to reverse or slow down maladaptive ventricular remodeling in these patients.[20] [8] Cooper LTJr, Keren A, Sliva K, et al. The global burden of myocarditis. Part 1: A systematic literature review for the global burden of diseases, injuries, and risk factors 2010 study. Global Heart 2014;9:121–9. [9] Liu PP, Mason JW. Advances in the understanding of myocarditis. Circulation 2001;104:275–80. [10] Okura Y, Dec GW, Hare JM, et al. A clinical and histopathologic comparison of cardiac sarcoidosis and idiopathic giant cell myocarditis. J Am Coll Cardiol 2003;41:322–5. [11] Dello Ruso A, Casella M, Pieroni M, et al. Drug-refractory ventricular tachycardia after myocarditis: endocardial and epicardial radiofrequency catheter ablation. Circ Arrhythm Electrophysiol 2012;5:492–8. [12] Saito J, Niwano S, Niwano H, et al. Electrical remodelling of the ventricular myocardium in myocarditis: studies of rat experimental autoimmune myocarditis. Circ J 2002;66:97–103. [13] Pieroni M, Smaldone C, Bellocci F. Cihakova D. Myocarditis presenting with ventricular arrhythmias: Role of electroanatomical mapping-guided endomyocardial biopsy in differential diagnosis. Myocarditis 2011;InTech, ISBN: 978-953-307-289-0, Available from: http://www.intechopen.com/ books/myocarditis/myocarditis-presenting-with-ventricular-arrhythmias- role-of-electroanatomical-mapping-guided-endomyo [published online 19 October 2011] References [1] Gore I, Saphir O. Myocarditis: a classiﬁcation of 1402 cases. Am Heart J 1947;34:827–30. [2] Anzini M, Merlo M, Sabbadini G, et al. Long-term evolution and prognostic stratiﬁcation of biopsy-proven active myocarditis. Circulation 2014;128:2384–94. [3] Kawai C. From myocarditis to cardiomyopathy: mechanisms of inﬂammation and cell death. Circulation 1999;99:1091–100. The combination of a triggering event and a susceptible myocardium has been evolving as a fundamental electrophysio- logical concept for the mechanism of the initiation of a potentially lethal arrhythmia. However, much is still to be elucidated from the interaction of the long-term risk of ventricular tachycardia in myocarditis and these independent risk factors. [4] D’Ambrosio A, Patti G, Manzoli A, et al. The fate of acute myocarditis between spontaneous improvement and evolution to dilated cardiomy- opathy: a review. Heart 2011;85:499–504. [5] McCarthy REIII, Boehmer JP, Hruban RH, et al. Long-term outcome of fulminant myocarditis as compared with acute (nonfulminant) myocar- ditis. N Engl J Med 2000;342:690–5. [6] Grogan M, Redﬁeld MM, Bailey KR, et al. Long-term outcome of patients with biopsy-proved myocarditis: comparison with idiopathic dilated cardiomyopathy. J Am Coll Cardiol 1995;26:80–4. 5. Conclusion To the best of our knowledge, the present study represents the longest follow-up period in patients with a history of myocarditis. The results of this study suggest a higher incidence of life- threatening VT and mortality during a very long-term follow-up in patients with a history of acute myocarditis. It also suggests that ACEis, ARBs, and BBs may play a protective role in preventing future VT events in these patients. Therefore, future work should focus on an in-depth risk stratiﬁcation of VT in myocarditis patients. Medicine Previous studies have demonstrated the role of a pharmacologic treatment, particularly with ACEis, ARBs, aldosterone antagonists, and BBs, in reversing or slowing down the maladaptive ventricular remodeling,[20] as well as their role in the prevention and treatment of ventricular arrhythmias. This may explain the protective role of these medications in our study patients. 4.4. Predictors of VTs Furthermore, our study showed that an older age, male gender, presence of DM, CKD, hyperlipidemia, and lesser use of ACEis, ARBs, and BBs were identiﬁed as independent risk factors for developing new-onset VT in patients with a history of myocarditis in the long-term follow-up. In our study, 55% of patients with a history of myocarditis who had VT were men. Although the exact physiologic mechanism that triggers this phenomenon is not clear, 5 Te et al. Medicine (2017) 96:18 Medicine 4.6. Study limitations The present study showed the long-term survival difference between patients with a history of myocarditis and the healthy population without structural heart disease. Nonetheless, there [14] Lampert R, McPherson CA, Clancy JF, et al. Gender differences in ventricular arrhythmia recurrence in patients with coronary artery 6 Te et al. Medicine (2017) 96:18 www.md-journal.com www.md-journal.com disease and implantable cardioverter-deﬁbrillators. J Am Coll CArdiol 2004;43:2293–9. [18] Chawla V, Greene T, Beck GJ, et al. Hyperlipidemia and long-term outcomes in nondiabetic chronic kidney disease. Clin J Am Soc Nephrol 2010;5:1582–7. [15] Rivero A, Curtis AB. Sex differences in arrhythmias. Curr Opin Cardiol 2010;25:8–15. [19] Frigerio M, Roubina E. Drugs for left ventricular remodeling in heart failure. Am J Cardiol 2005;96(suppl):10–8. [16] Vinik AI, Ziegler D. Diabetic cardiovascular autonomic neuropathy. Circulation 2007;115:387–97. [20] Kostam MA, Kronenberg MW, Rousseau MF, et al. Effects of the angiotensin-converting enzyme inhibitor, enalapril, on the long term progression of left ventricular dilatation in patients with asymptomatic systolic dysfunction. Circulation 1993;88:2277–83. [17] Bonato FOB, Lemos MM, Cassiolato JL, et al. Prevalence of ventricular arrhythmia and its associated factors in nondialyzed chronic kidney disease patients. PLoS One 2013;8:e66036. 7 7 7
https://openalex.org/W1971904580	https://europepmc.org/articles/pmc4402570?pdf=render	English	null	A New Approach to Define and Diagnose Cardiometabolic Disorder in Children	Journal of diabetes research	2,015	cc-by	9,839	Lars Bo Andersen,1,2 Jeppe Bo Lauersen,2 Jan Christian Brønd,1 Sigmund Alfred Anderssen,2 Luis B. Sardinha,3 Jostein Steene-Johannessen,2 Robert G. McMurray,4 Mauro V. G. Barros,5 Susi Kriemler,6 Niels Christian Møller,1 Anna Bugge,1 Peter Lund Kristensen,1 Mathias Ried-Larsen,1,7 Anders Grøntved,1 and Ulf Ekelund2,8 Lars Bo Andersen,1,2 Jeppe Bo Lauersen,2 Jan Christian Brønd,1 Sigmund Alfred Anderssen,2 Luis B. Sardinha,3 Jostein Steene-Johannessen,2 Robert G. McMurray,4 Mauro V. G. Barros,5 Susi Kriemler,6 Niels Christian Møller,1 Anna Bugge,1 Peter Lund Kristensen,1 Mathias Ried-Larsen,1,7 Anders Grøntved,1 and Ulf Ekelund2,8 2Department of Sports Medicine, Norwegian School of Sport Sciences, Sognsveien 220, 0806 Oslo, Norway 2Department of Sports Medicine, Norwegian School of Sport Sciences, Sognsveien 220, 0806 Oslo, Norway 3Exercise and Health Laboratory, CIPER, Fac Motricidade Humana, Universidade de Lisboa, 2Department of Sports Medicine, Norwegian School of Sport Sciences, Sognsveien 220, 0806 Oslo, Norway 3 Department of Sports Medicine, Norwegian School of Sport Sciences, Sognsveien 220, 0806 Oslo, Norway 3Exercise and Health Laboratory, CIPER, Fac Motricidade Humana, Universidade de Lisboa, Estrada Dacosth, Cruz-Quebrada, 1499 Lisbon, Portugal 3Exercise and Health Laboratory, CIPER, Fac Motricidade Humana, Universidade de Lisboa, Estrada Dacosth, Cruz-Quebrada, 1499 Lisbon, Portugal g Department of Exercise and Sport Science, University of North Carolina, 025 Fetzer Gym, CB No. 8700, Chapel Hill, NC 27599-8700, USA 5School of Physical Education, University of Pernambuco, Campus Universitario HUOC-ESEF, Arnobio Marques 310, Santo Amaro, 50.100-130 Recife, PE, Brazil 5School of Physical Education, University of Pernambuco, Campus Universitario HUOC-ESEF, Arnobio Marques 310, Santo Amaro, 50.100-130 Recife, PE, Brazil q f 6Epidemiology, Biostatistics and Prevention Institute, University of Z¨urich, Hirschengraben 84, 8001 Z¨urich, Switzerland 7The Centre of Inflammation and Metabolism and Trygfondens Center for Aktiv Sundhed, Department of Infectious Diseases and CMRC, Rigshospitalet, Faculty of Health Sciences, University of Copenhagen, Tagensvej 20, 2100 Copenhagen, Denmark 8MRC Epidemiology Unit, Institute of Metabolic Science, Addenbrooke’s Hospital, University of Cambridge, Hills Road, Cambridge CB2 0QQ, UK Correspondence should be addressed to Lars Bo Andersen; lboandersen@health.sdu.dk Received 26 November 2014; Accepted 17 March 2015 Received 26 November 2014; Accepted 17 March 2015 Received 26 November 2014; Accepted 17 March 2015 Academic Editor: Francesco Chiarelli Academic Editor: Francesco Chiarelli Copyright © 2015 Lars Bo Andersen 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. The aim of the study was to test the performance of a new definition of metabolic syndrome (MetS), which better describes metabolic dysfunction in children. Methods. 15,794 youths aged 6–18 years participated. Mean z-score for CVD risk factors was calculated. Lars Bo Andersen,1,2 Jeppe Bo Lauersen,2 Jan Christian Brønd,1 Sigmund Alfred Anderssen,2 Luis B. Sardinha,3 Jostein Steene-Johannessen,2 Robert G. McMurray,4 Mauro V. G. Barros,5 Susi Kriemler,6 Niels Christian Møller,1 Anna Bugge,1 Peter Lund Kristensen,1 Mathias Ried-Larsen,1,7 Anders Grøntved,1 and Ulf Ekelund2,8 Sensitivity analyses were performed to evaluate which parameters best described the metabolic dysfunction by analysing the score against independent variables not included in the score. Results. More youth had clustering of CVD risk factors (>6.2%) compared to the number selected by existing MetS definitions (International Diabetes Federation (IDF) < 1%). Waist circumference and BMI were interchangeable, but using insulin resistance homeostasis model assessment (HOMA) instead of fasting glucose increased the score. The continuous MetS score was increased when cardiorespiratory fitness (CRF) and leptin were included. A mean z-score of 0.40–0.85 indicated borderline and above 0.85 indicated clustering of risk factors. A noninvasive risk score based on adiposity and CRF showed sensitivity and specificity of 0.85 and an area under the curve of 0.92 against IDF definition of MetS. Conclusions. Diagnosis for MetS in youth can be improved by using continuous variables for risk factors and by including CRF and leptin. Hindawi Publishing Corporation Journal of Diabetes Research Volume 2015, Article ID 539835, 10 pages http://dx.doi.org/10.1155/2015/539835 Hindawi Publishing Corporation Journal of Diabetes Research Volume 2015, Article ID 539835, 10 pages http://dx.doi.org/10.1155/2015/539835 Hindawi Publishing Corporation Journal of Diabetes Research Volume 2015, Article ID 539835, 10 pages http://dx.doi.org/10.1155/2015/539835 1. Introduction links several apparently unrelated biological events into a sin- gle pathophysiological assemble; that is, several cardiovascu- lar disease (CVD) risk factors seemed to be increased simul- taneously in some individuals. Despite different definitions Metabolic syndrome (MetS) was first described by Reaven in the mid 1980s [1]. MetS is a conceptual framework, which 2 Journal of Diabetes Research 2 of summed z-scores, which selected the same number of children as calculated above, where risk factors were not independently distributed; (d) to construct a program where absolute values of the risk factors can be entered, and the program calculates the mean z-score of the included variables (age and sex adjusted) to evaluate if the child has a disorder; (e) to do sensitivity analyses to evaluate if other risk factors may improve diagnostic criteria; and (f) if it matters to substitute a measure of a certain trait with another such as including z-score of body mass index (BMI) instead of waist circumference. The program makes it easy for the general practitioner to use the diagnostic tool. Last, we tested a noninvasive measure of metabolic risk based on physical fitness and fatness.h most agree on the individual components constituting the MetS, which include dyslipidemia (triglycerides and choles- terol), hypertension, glucose intolerance, and adiposity [2–5]. Originally, the different CVD risk factors were mainly treated with drugs and it therefore seemed logical to use the cutoff point for each risk factor in the definition of the criteria for MetS. Criteria have been suggested by several organizations and researchers and have changed over time even if they all build on the same concept. Insulin resistance triggered a common mechanism affecting blood pressure (BP), high density lipoprotein cholesterol (HDL), triglycerides (TG), and glucose tolerance, and central obesity may cause the insulin resistance and therefore be a central part of MetS. This concept subsequently evolved to encompass a number of multiple definitions [2] established by the WHO [3, 5], the National Cholesterol Education Program Adult Treatment Panel III (ATP III) [4], and the International Diabetes Federation (IDF) [5]. Modified criteria based on the same concept as in adults have been suggested in children [6–9].i i This approach will make it possible to use the available information and get a better measure of the risk of the child. Information is not reduced when we use continuous variables and the composite z-score can be used as a continuous or dichotomous variable. 1. Introduction It is hypothesized that the z-score of the different variables covering the same trait will only differ slightly; that is, the mean z-score only changes marginally if BMI is entered instead of waist, which makes the program flexible and enables comparison between studies using dif- ferent measures. Further, this approach makes it possible to include other risk factors in the calculation of mean z-score or exclude them if data is not available. The constructed program is available on the Internet or as an APP for smartphone, and the general practitioner can enter the available information, which should include as many of the risk factors as possible. Existing MetS definitions have shortcomings, especially for children, and because studies use different definitions comparison between studies is difficult. All definitions are based on dichotomisation of the CVD risk factors and to be clinically diagnosed with the MetS the thresholds for at least three risk factors including obesity must be attained. Limitations include (1) reduction of available information of risk by dichotomizing variables; (2) different risk factors that are given different weight (i.e., prevalence of the risk factors differs, which means that few are selected based on the rare risk factors); (3) thresholds for the individual risk factors that are arbitrarily chosen in children, where no hard endpoints exist; (4) selection of risk factors that exclude potentially important variables; for example, the use of fasting glucose in children rather than fasting insulin or HOMA score as mea- sure of impaired glucose regulation may conceal important information; many children with severe insulin resistance are still able to regulate their fasting blood glucose well [10]; (5) different definitions that use different blood variables and fatness variables. This problem makes it difficult to compare prevalence between populations. These shortcomings result in substantial misclassification, and comparison between studies using different criteria is difficult. When children are analysed, there is also a major difference between the number of children diagnosed with MetS compared to the number where a clustering of risk factors occurs [11, 12]. (BMI) or skinfold instead of waist circumference or HOMA instead of fasting glucose. (BMI) or skinfold instead of waist circumference or HOMA instead of fasting glucose. (BMI) or skinfold instead of waist circumference or HOMA instead of fasting glucose. (BMI) or skinfold instead of waist circumference or HOMA instead of fasting glucose. 2.2. Analytical Process and Statistics. Statistical analysis was performed in IBM SPSS Statistics 22 and Stata 12 (meta- analysis).f (5) Fifth step is sensitivity analyses to evaluate if addition of other risk factors improved diagnostic criteria. For this purpose we examined the associations between CRF (independent variable) by logistic regression and calculated odds ratio (OR) for the three lower quar- tiles of CRF against the MetS variables (dependent variable). Additional risk factors (APOA1, APOB, leptin, and CRP) beyond those included in the IDF criteria were tested to examine if their inclusion strengthened the magnitude of association between CRF and the mean z-score of the traditional MetS risk factors. In all analyses a threshold in the mean z-score, identifying the same proportion of children, was identified. Inclusion of CRF in the mean z-score was tested by removing waist circumference from the MetS and using it as independent variable to see if the association became stronger when fitness was included in the MetS outcome. (5) Fifth step is sensitivity analyses to evaluate if addition of other risk factors improved diagnostic criteria. For this purpose we examined the associations between CRF (independent variable) by logistic regression and calculated odds ratio (OR) for the three lower quar- tiles of CRF against the MetS variables (dependent variable). Additional risk factors (APOA1, APOB, leptin, and CRP) beyond those included in the IDF criteria were tested to examine if their inclusion strengthened the magnitude of association between CRF and the mean z-score of the traditional MetS risk factors. In all analyses a threshold in the mean z-score, identifying the same proportion of children, was identified. Inclusion of CRF in the mean z-score was tested by removing waist circumference from the MetS and using it as independent variable to see if the association became stronger when fitness was included in the MetS outcome. y Common estimates were calculated using random effect meta-analysis [13]. Analysis was progressed by the following steps. (1) First step is calculation of the number of chil- dren where risk factors were not independently dis- tributed; that is, risk factors clustered. MetS risk factors follow a binomial distribution if they are independently distributed. (BMI) or skinfold instead of waist circumference or HOMA instead of fasting glucose. 𝑁is the total number of children in the cohort and 𝑛𝑖is the number of children with the specific number of risk factors. (6) Prognostic value of a MetS outcome including only noninvasive variables (waist/height and inverse CRF) was also tested. We used waist circumfer- ence/height as a proxy for fatness, because this variable is independent of age [20]. The mean z- score of ((waist/height) + (1/CRF))/2 was examined in a receiver operating characteristic (ROC) analysis against the IDF definition of the MetS and the clus- tered MetS score variable constructed from z-scores of log TG, log waist, systolic BP, log HOMA, inverse HDL, and inverse fitness. (6) Prognostic value of a MetS outcome including only noninvasive variables (waist/height and inverse CRF) was also tested. We used waist circumfer- ence/height as a proxy for fatness, because this variable is independent of age [20]. The mean z- score of ((waist/height) + (1/CRF))/2 was examined in a receiver operating characteristic (ROC) analysis against the IDF definition of the MetS and the clus- tered MetS score variable constructed from z-scores of log TG, log waist, systolic BP, log HOMA, inverse HDL, and inverse fitness. All analytical steps were built into an Internet-based pro- gram which calculates MetS score in order to facilitate the use in general practice (http://www.video4coach.com/zscore/ index.html).h (2) The association between age and each risk factor was calculated using linear regression in order to compute age adjusted risk factor levels for each child. All risk factors were adjusted to the age of 12 years for each gender to enable comparison of levels between cohorts of different age by the following formula: risk factoradjusted = risk factor −(𝛽∗(age −12)). BMI, waist circumference, sum of 4 skinfolds, TG, glucose, HOMA, C-reactive protein (CRP), APOA1, APOB, and leptin were skewed and therefore log- transformed before z-scores were constructed. z- scores were constructed using mean and SD of the age adjusted risk factors for the total sample. The mean of the 5 risk factors included in the IDF definition of MetS [6] was calculated using HOMA rather than fasting glucose as an indicator of glucose metabolism. The proportion of children categorised as having the metabolic syndrome (IDF definition) was calculated accord- ing to Zimmet et al. [6], where age- and sex-specific 90th percentiles for waist circumference were defined according to Fern´andez et al. [21]. (BMI) or skinfold instead of waist circumference or HOMA instead of fasting glucose. If the observed number of children with a given number of risk factors exceeds the expected, risk factors exhibit clustering. For this analysis we defined the upper quartile of waist circumference, systolic blood pressure (BP), triglyceride (TG), and HOMA together with the lower quartile of high density lipoprotein cholesterol (HDL) to be at risk for each cohort separately. Proportion of expected subjects with a specific number of risk fac- tors according to the binomial distribution was (𝑛! ∗ 𝑝𝑟∗(1 −𝑝)𝑛−𝑟)/(𝑟! ∗(𝑛−𝑟)!), where 𝑛is the possible number of risk factors (5), 𝑝is the proportion having the risk factor (25%), and 𝑟is the number of risk factors the probability is calculated for. The expected proportions of children having 0 to 5 risk factors were 0.178, 0.356, 0.297, 0.132, 0.057, 0.0044, and 0.0002, respectively. We then divided the observed number of children with a specific number of risk factors with the expected and calculated 95% confidence intervals as exp(Ln(OR) + / −1.95 ∗SE(Ln(OR))), where SE(Ln(OR) = SQRT(1/𝑛𝑖) + (1/(𝑁−𝑛𝑖))). 𝑁is the total number of children in the cohort and 𝑛𝑖is the number of children with the specific number of risk factors. (1) First step is calculation of the number of chil- dren where risk factors were not independently dis- tributed; that is, risk factors clustered. MetS risk factors follow a binomial distribution if they are independently distributed. If the observed number of children with a given number of risk factors exceeds the expected, risk factors exhibit clustering. For this analysis we defined the upper quartile of waist circumference, systolic blood pressure (BP), triglyceride (TG), and HOMA together with the lower quartile of high density lipoprotein cholesterol (HDL) to be at risk for each cohort separately. Proportion of expected subjects with a specific number of risk fac- tors according to the binomial distribution was (𝑛! ∗ 𝑝𝑟∗(1 −𝑝)𝑛−𝑟)/(𝑟! ∗(𝑛−𝑟)!), where 𝑛is the possible number of risk factors (5), 𝑝is the proportion having the risk factor (25%), and 𝑟is the number of risk factors the probability is calculated for. The expected proportions of children having 0 to 5 risk factors were 0.178, 0.356, 0.297, 0.132, 0.057, 0.0044, and 0.0002, respectively. We then divided the observed number of children with a specific number of risk factors with the expected and calculated 95% confidence intervals as exp(Ln(OR) + / −1.95 ∗SE(Ln(OR))), where SE(Ln(OR) = SQRT(1/𝑛𝑖) + (1/(𝑁−𝑛𝑖))). 2. Methods 2.1. Participants. We pooled cross-sectional data from 23 population based cohorts in children and adolescents aged 6–18 years. The 23 cohorts were merged into 18 cohorts before analysis. Ten cohorts (𝑁= 4806) of 9- and 15-year- old boys and girls from the European Youth Heart Study (EYHS) (1997 through 2007) were included. Nine-year-olds and 15-year-olds, respectively, assessed at two different time points from the same country were pooled before analysis. Additionally, four cohorts of 9- and 15-year-olds from EYHS Norway (𝑁= 3020) were included but analysed as four separate cohorts, as two cohorts lacked important blood variables. The Copenhagen School Child Intervention Study (CoSCIS) (𝑁= 1816) included three measurements collected in 2001, 2004, and 2008. Data from the US NHANES (𝑁= 4801) collected in 2003 and 2005 were merged and recoded into three age groups (<10 years, 10–14 years, and >14 years). Finally, children from Switzerland (KISS) were sampled from 1st (mean age 8 years) and 5th grade school children (mean age 12 years) and were measured three times over two years (𝑁 = 1351). Successive measurements of a child were regarded as multiple individuals. In total 7902 girls and 7892 boys were included in the analysis. Data collection procedures and analytical methods have previously been described in detail (EYHS [13], CoSCIS [14, 15], KISS [16], NHANES [17], Norwegian EYHS 2000 [18], and Norwegian EYHS 2005 [19]). Ethical approval has been obtained for all included cohort studies in their respective countries. The current definitions in use preclude accurate esti- mates of the MetS prevalence between populations and the dichotomisation of variables may attenuate or preclude real associations between lifestyle behaviours and metabolic risk.h The aim of this study was to develop a novel method for identifying young people with increased metabolic risk and solve weaknesses of former definitions. Furthermore, we tested a simple noninvasive screening tool to identify children where further investigation is indicated. The steps in the analysis were (a) to calculate the number of children where risk factors were not independently distributed; (b) to construct age adjusted z-scores for each risk factor based on common means and SDs for the whole database for genders separately; (c) to define a cutoff point in mean 3 Journal of Diabetes Research 3. Results Girls Waist (cm) Systolic BP (mmHg) HDL (mmol/L) Triglyceride (mmol/L) HOMA 𝑁 Mean SD 𝑁 Mean SD 𝑁 Mean SD 𝑁 Mean SD 𝑁 Mean SD Denmark 9 yr (EYHS) 547 18.9 2.6 547 104.4 8.2 547 1.47 0.31 547 0.88 0.38 547 2.00 .97 Denmark 15 yr (EYHS) 464 18.6 2.9 464 102.2 8.5 464 1.52 0.32 464 0.86 0.40 464 1.76 1.13 Portugal 9 yr (EYHS) 501 20.1 3.4 501 98.1 8.9 501 1.52 0.31 501 0.82 0.38 501 1.61 .87 Portugal 15 yr (EYHS) 378 19.1 3.2 378 96.3 8.2 378 1.56 0.31 378 0.63 0.35 378 1.29 1.02 Estonia 9 yr (EYHS) 303 18.2 2.3 303 103.8 8.9 303 1.40 0.29 303 0.83 0.28 303 1.71 .78 Estonia 15 yr (EYHS) 333 18.0 2.8 333 102.6 9.2 333 1.50 0.28 333 0.77 0.36 333 1.96 1.23 Norway 9 yr 2000 (EYHS) 196 18.7 2.4 196 105.5 8.3 196 1.45 0.30 196 0.97 0.30 ⋅ ⋅ Norway 15 yr 2000 (EYHS) 181 17.8 3.2 181 103.8 8.8 181 1.42 0.31 181 0.98 0.47 ⋅ ⋅ Denmark 6 yr (CoSCIS) 333 19.7 1.9 333 104.9 7.7 333 1.38 0.27 333 0.74 0.29 333 1.54 .45 Denmark 9 yr (CoSCIS) 338 19.0 2.6 338 106.4 8.9 338 1.54 0.37 338 0.63 0.24 338 1.64 .75 Denmark 13 yr (CoSCIS) 252 18.4 2.8 252 106.9 7.9 252 1.50 0.33 252 0.76 0.37 252 2.06 1.23 USA 6–10 yr (NHANES) 576 20.6 3.7 576 104.9 8.8 576 1.40 0.33 576 1.06 0.61 ⋅ ⋅ USA 10–14 yr (NHANES) 796 20.9 5.2 796 106.4 9.2 796 1.45 0.36 796 0.90 0.48 796 2.51 3.67 USA 14–18 yr (NHANES) 1046 21.0 5.8 1046 107.7 10.0 1046 1.38 0.30 1046 0.90 0.90 1046 1.91 3.37 Norway 9 yr 2005 (EYHS) 604 19.2 2.7 604 105.9 7.7 604 1.66 0.35 604 0.77 0.33 604 1.49 .71 Norway 15 yr 2005 (EYHS) 479 18.7 2.9 479 104.2 8.8 479 1.68 0.34 479 0.71 0.32 479 1.26 1.19 Switzerland 8 yr (KISS) 320 19.2 2.4 320 105.9 8.1 320 1.50 0.36 320 0.72 0.29 320 1.54 .66 Switzerland 12 yr (KISS) 345 18.5 2.9 345 107.5 8.8 345 1.62 0.37 345 0.64 0.32 345 1.46 .82 Table 1: Description of age adjusted risk factor levels for all cohorts. Adjustment for age enables comparison between cohorts. 3. Results Age, anthropometrics, and CVD risk factors are described for each cohort in web-appendix (see web-only Table A in Supplementary Material available online at http://dx.doi.org/ 10.1155/2015/539835). Characteristics of CVD risk factors are also described after adjustment for age to enable comparison of levels between cohorts of different age (Table 1). The association between age and each risk factor is shown in Table 2.h (3) Third step is definition of a cut-point in mean of summed z-scores, which selected the number of children with clustered CVD risk as calculated in step (1). (3) Third step is definition of a cut-point in mean of summed z-scores, which selected the number of children with clustered CVD risk as calculated in step (1). The number of children according to number of risk factors defined as the upper quartile (0–5 risk factors), the corresponding mean z-score, and the proportion defined as having MetS according to IDF definition are shown in Table 3. Figure 1 and web-only Figures A–D show in which cohorts and groups risk factors cluster, that is, ORs for observed (4) Fourth step is determination of the significance of substituting a measure of a certain trait with another, for example, including z-score of body mass index (4) Fourth step is determination of the significance of substituting a measure of a certain trait with another, for example, including z-score of body mass index 4 Journal of Diabetes Research Table 1: Description of age adjusted risk factor levels for all cohorts. Adjustment for age enables comparison between cohorts. 3. Results Girls Waist (cm) Systolic BP (mmHg) HDL (mmol/L) Triglyceride (mmol/L) HOMA 𝑁 Mean SD 𝑁 Mean SD 𝑁 Mean SD 𝑁 Mean SD 𝑁 Mean SD Denmark 9 yr (EYHS) 547 18.9 2.6 547 104.4 8.2 547 1.47 0.31 547 0.88 0.38 547 2.00 .97 Denmark 15 yr (EYHS) 464 18.6 2.9 464 102.2 8.5 464 1.52 0.32 464 0.86 0.40 464 1.76 1.13 Portugal 9 yr (EYHS) 501 20.1 3.4 501 98.1 8.9 501 1.52 0.31 501 0.82 0.38 501 1.61 .87 Portugal 15 yr (EYHS) 378 19.1 3.2 378 96.3 8.2 378 1.56 0.31 378 0.63 0.35 378 1.29 1.02 Estonia 9 yr (EYHS) 303 18.2 2.3 303 103.8 8.9 303 1.40 0.29 303 0.83 0.28 303 1.71 .78 Estonia 15 yr (EYHS) 333 18.0 2.8 333 102.6 9.2 333 1.50 0.28 333 0.77 0.36 333 1.96 1.23 Norway 9 yr 2000 (EYHS) 196 18.7 2.4 196 105.5 8.3 196 1.45 0.30 196 0.97 0.30 ⋅ ⋅ Norway 15 yr 2000 (EYHS) 181 17.8 3.2 181 103.8 8.8 181 1.42 0.31 181 0.98 0.47 ⋅ ⋅ Denmark 6 yr (CoSCIS) 333 19.7 1.9 333 104.9 7.7 333 1.38 0.27 333 0.74 0.29 333 1.54 .45 Denmark 9 yr (CoSCIS) 338 19.0 2.6 338 106.4 8.9 338 1.54 0.37 338 0.63 0.24 338 1.64 .75 Denmark 13 yr (CoSCIS) 252 18.4 2.8 252 106.9 7.9 252 1.50 0.33 252 0.76 0.37 252 2.06 1.23 USA 6–10 yr (NHANES) 576 20.6 3.7 576 104.9 8.8 576 1.40 0.33 576 1.06 0.61 ⋅ ⋅ USA 10–14 yr (NHANES) 796 20.9 5.2 796 106.4 9.2 796 1.45 0.36 796 0.90 0.48 796 2.51 3.67 USA 14–18 yr (NHANES) 1046 21.0 5.8 1046 107.7 10.0 1046 1.38 0.30 1046 0.90 0.90 1046 1.91 3.37 Norway 9 yr 2005 (EYHS) 604 19.2 2.7 604 105.9 7.7 604 1.66 0.35 604 0.77 0.33 604 1.49 .71 Norway 15 yr 2005 (EYHS) 479 18.7 2.9 479 104.2 8.8 479 1.68 0.34 479 0.71 0.32 479 1.26 1.19 Switzerland 8 yr (KISS) 320 19.2 2.4 320 105.9 8.1 320 1.50 0.36 320 0.72 0.29 320 1.54 .66 Switzerland 12 yr (KISS) 345 18.5 2.9 345 107.5 8.8 345 1.62 0.37 345 0.64 0.32 345 1.46 .82 Boys Waist (cm) Systolic BP (mmHg) HDL (mmol/L) Triglyceride (mmol/L) HOMA 𝑁 Mean SD 𝑁 Mean SD 𝑁 Mean SD 𝑁 Mean SD 𝑁 Mean SD Denmark 9 yr (EYHS) 459 18.9 2.5 459 106.6 7.86 459 1.55 0.37 459 0.78 0.34 459 1.79 .94 Denmark 15 yr (EYHS) 391 18.4 2.7 391 109.4 10.99 391 1.41 0.28 391 0.79 0.48 391 1.83 1.64 Portugal 9 yr (EYHS) 538 20.1 3.3 538 99.0 8.59 538 1.55 0.32 538 0.74 0.35 538 1.33 .58 Portugal 15 yr (EYHS) 358 18.5 3.1 358 102.3 10.63 358 1.40 0.27 358 0.60 0.31 358 .91 .93 Estonia 9 yr (EYHS) 278 18.3 1.9 278 105.2 10.59 278 1.44 0.32 278 0.75 0.31 278 1.59 .68 Estonia 15 yr (EYHS) 260 17.9 2.6 260 109.8 11.97 260 1.39 0.27 260 0.65 0.32 260 1.80 1.58 Norway 9 yr 2000 (EYHS) 210 18.6 2.0 210 107.0 6.73 210 1.60 0.41 210 0.87 0.34 ⋅ ⋅ ⋅ Norway 15 yr 2000 (EYHS) 167 17.9 2.4 167 111.0 9.95 167 1.31 0.31 167 1.02 0.58 ⋅ ⋅ ⋅ Denmark 6 yr (CoSCIS) 373 19.6 1.7 373 105.9 7.79 373 1.36 0.26 373 0.71 0.22 373 1.55 .65 Denmark 9 yr (CoSCIS) 368 19.0 2.5 368 108.6 8.73 368 1.54 0.32 368 0.60 0.26 368 1.53 .76 Denmark 13 yr (CoSCIS) 259 18.1 2.6 259 109.4 9.10 259 1.46 0.28 259 0.74 0.38 259 1.80 1.20 USA 6–10 yr (NHANES) 620 20.8 3.7 620 105.3 9.36 620 1.33 0.30 620 1.04 0.49 ⋅ ⋅ ⋅ USA 10–14 yr (NHANES) 775 22.2 5.7 775 104.9 9.77 775 1.40 0.33 775 1.02 0.54 775 2.92 2.18 USA 14–18 yr (NHANES) 988 21.6 6.2 988 101.5 9.49 988 1.55 0.34 988 0.81 0.52 988 2.00 3.42 Norway 9 yr 2005 702 18.9 2.5 702 106.8 7.71 702 1.73 0.40 702 0.69 0.32 702 1.41 .67 Norway 15 yr 2005 514 18.4 3.4 514 110.1 8.95 514 1.51 0.30 514 0.73 0.48 514 1.26 1.29 Switzerland 8 yr (KISS) 337 19.2 2.6 337 106.5 9.06 337 1.51 0.33 337 0.75 0.28 337 1.67 .75 Switzerland 12 yr (KISS) 390 18.5 2.6 390 105.4 8.69 390 1.61 0.35 390 0.68 0.30 390 1.58 .95 y ( ) Boys Waist (cm) Systolic BP (mmHg) HDL (mmol/L) Triglyceride (mmol/L) HOMA 𝑁 Mean SD 𝑁 Mean SD 𝑁 Mean SD 𝑁 Mean SD 𝑁 Mean SD Denmark 9 yr (EYHS) 459 18.9 2.5 459 106.6 7.86 459 1.55 0.37 459 0.78 0.34 459 1.79 .94 Denmark 15 yr (EYHS) 391 18.4 2.7 391 109.4 10.99 391 1.41 0.28 391 0.79 0.48 391 1.83 1.64 Portugal 9 yr (EYHS) 538 20.1 3.3 538 99.0 8.59 538 1.55 0.32 538 0.74 0.35 538 1.33 .58 Portugal 15 yr (EYHS) 358 18.5 3.1 358 102.3 10.63 358 1.40 0.27 358 0.60 0.31 358 .91 .93 Estonia 9 yr (EYHS) 278 18.3 1.9 278 105.2 10.59 278 1.44 0.32 278 0.75 0.31 278 1.59 .68 Estonia 15 yr (EYHS) 260 17.9 2.6 260 109.8 11.97 260 1.39 0.27 260 0.65 0.32 260 1.80 1.58 Norway 9 yr 2000 (EYHS) 210 18.6 2.0 210 107.0 6.73 210 1.60 0.41 210 0.87 0.34 ⋅ ⋅ ⋅ Norway 15 yr 2000 (EYHS) 167 17.9 2.4 167 111.0 9.95 167 1.31 0.31 167 1.02 0.58 ⋅ ⋅ ⋅ Denmark 6 yr (CoSCIS) 373 19.6 1.7 373 105.9 7.79 373 1.36 0.26 373 0.71 0.22 373 1.55 .65 Denmark 9 yr (CoSCIS) 368 19.0 2.5 368 108.6 8.73 368 1.54 0.32 368 0.60 0.26 368 1.53 .76 Denmark 13 yr (CoSCIS) 259 18.1 2.6 259 109.4 9.10 259 1.46 0.28 259 0.74 0.38 259 1.80 1.20 USA 6–10 yr (NHANES) 620 20.8 3.7 620 105.3 9.36 620 1.33 0.30 620 1.04 0.49 ⋅ ⋅ ⋅ USA 10–14 yr (NHANES) 775 22.2 5.7 775 104.9 9.77 775 1.40 0.33 775 1.02 0.54 775 2.92 2.18 USA 14–18 yr (NHANES) 988 21.6 6.2 988 101.5 9.49 988 1.55 0.34 988 0.81 0.52 988 2.00 3.42 Norway 9 yr 2005 702 18.9 2.5 702 106.8 7.71 702 1.73 0.40 702 0.69 0.32 702 1.41 .67 Norway 15 yr 2005 514 18.4 3.4 514 110.1 8.95 514 1.51 0.30 514 0.73 0.48 514 1.26 1.29 Switzerland 8 yr (KISS) 337 19.2 2.6 337 106.5 9.06 337 1.51 0.33 337 0.75 0.28 337 1.67 .75 Switzerland 12 yr (KISS) 390 18.5 2.6 390 105.4 8.69 390 1.61 0.35 390 0.68 0.30 390 1.58 .95 Systolic BP (mmHg) M SD Characteristic Regression 𝛽for age Girls Boys Mean 𝛽 𝑃< Mean 𝛽 𝑃< BMI (kg/m2) 19.6 0.704 0.001 19.6 0.699 0.001 Waist circumference (cm) 67.4 2.190 0.001 68.3 2.317 0.001 Ratio waist to hip circumference 77.8 −0.014 0.001 75.6 0.012 0.001 Sum of 4 skinfolds (mm) 42.9 2.453 0.001 33.5 0.743 0.001 Diastolic BP (mmHg) 61.5 0.386 0.001 62.0 0.442 0.001 Systolic BP (mmHg) 105.2 1.339 0.001 106.1 1.382 0.001 Total cholesterol (mmol/L) 4.29 −0.032 0.001 4.10 −0.070 0.001 HDL-cholesterol (mmol/L) 1.48 −0.019 0.001 1.48 −0.025 0.001 LDL-cholesterol (mmol/L) 2.40 −0.039 0.001 2.29 −0.044 0.001 Triglyceride (mmol/L) 0.80 0.025 0.001 .75 0.028 0.001 Glucose (mmol/L) 4.99 0.041 0.001 5.06 0.042 0.001 Insulin (pmol/L) 55.0 4.476 0.001 51.1 4.522 0.001 CRF (VO2max [mL/min/kg]) 42.5 −0.634 0.001 50.5 0.418 0.001 CRP (mg/dL) .84 −0.015 NS .74 0.006 NS APOA1 (𝜇mol/L) 11.2 1.311 0.001 10.6 1.197 0.001 APOB (𝜇mol/L) 1.00 0.037 0.001 .94 0.032 0.001 Leptin (ng/mL) .93 0.099 0.001 .37 −0.007 NS Adiponectin (𝜇g/mL) 17.0 −0.396 0.001 15.2 −0.841 0.001 HOMA 12.6 1.111 0.001 11.8 1.129 0.001 Ln of OR for 4 + 5 RF 0.75 1 10 Combined Sw 12yr Sw 8yr N 15yr 2005 N 9yr 2005 US 14–18yr US 10–14yr Cop 13yr Cop 9yr Cop 6yr Est 15yr Est 9yr Mad 15yr Mad 9yr DK 15yr DK 9yr 3.1. Sensitivity Analyses (Table 4). Analyses of composite MetS score were compared when sum of 4 skinfolds or BMI were used rather than waist circumference as the adiposity risk factor. The Pearson correlation between the mean z-scores including either BMI, waist circumference, or skinfold, respectively, was >0.97 for all associations. When the associations between CRF and dichotomized variables of the three mean z-scores were examined, greater magnitude of association was found when sum of skinfolds was used as the adiposity variable in the composite score, but similar magni- tude was found for scores including waist circumference and BMI (Table 4). Sensitivity analysis was conducted to examine whether the magnitude of associations between CRF and the MetS score differed when HOMA was included in the score rather than fasting glucose, keeping other variables constant. The magnitude of association was greater when the MetS composite score included HOMA rather than fasting glucose (Table 4). Including APOA1, APOB, adiponectin, or CRP in the MetS score did not strengthen the association between CRF and MetS score even if they were associated with MetS score. Systolic BP (mmHg) M SD Sensitivity analysis was conducted to examine whether the magnitude of associations between CRF and the MetS score differed when HOMA was included in the score rather than fasting glucose, keeping other variables constant. The magnitude of association was greater when the MetS composite score included HOMA rather than fasting glucose Journal of Diabetes Research 5 Table 2: Overall mean value and regression 𝛽-values with risk factors and age. 𝛽-values were used to calculate age adjusted risk factor levels. Characteristic Regression 𝛽for age Girls Boys Mean 𝛽 𝑃< Mean 𝛽 𝑃< BMI (kg/m2) 19.6 0.704 0.001 19.6 0.699 0.001 Waist circumference (cm) 67.4 2.190 0.001 68.3 2.317 0.001 Ratio waist to hip circumference 77.8 −0.014 0.001 75.6 0.012 0.001 Sum of 4 skinfolds (mm) 42.9 2.453 0.001 33.5 0.743 0.001 Diastolic BP (mmHg) 61.5 0.386 0.001 62.0 0.442 0.001 Systolic BP (mmHg) 105.2 1.339 0.001 106.1 1.382 0.001 Total cholesterol (mmol/L) 4.29 −0.032 0.001 4.10 −0.070 0.001 HDL-cholesterol (mmol/L) 1.48 −0.019 0.001 1.48 −0.025 0.001 LDL-cholesterol (mmol/L) 2.40 −0.039 0.001 2.29 −0.044 0.001 Triglyceride (mmol/L) 0.80 0.025 0.001 .75 0.028 0.001 Glucose (mmol/L) 4.99 0.041 0.001 5.06 0.042 0.001 Insulin (pmol/L) 55.0 4.476 0.001 51.1 4.522 0.001 CRF (VO2max [mL/min/kg]) 42.5 −0.634 0.001 50.5 0.418 0.001 CRP (mg/dL) .84 −0.015 NS .74 0.006 NS APOA1 (𝜇mol/L) 11.2 1.311 0.001 10.6 1.197 0.001 APOB (𝜇mol/L) 1.00 0.037 0.001 .94 0.032 0.001 Leptin (ng/mL) .93 0.099 0.001 .37 −0.007 NS Adiponectin (𝜇g/mL) 17.0 −0.396 0.001 15.2 −0.841 0.001 HOMA 12.6 1.111 0.001 11.8 1.129 0.001 Ln of OR for 4 + 5 RF 0.75 1 10 Combined Sw 12yr Sw 8yr N 15yr 2005 N 9yr 2005 US 14–18yr US 10–14yr Cop 13yr Cop 9yr Cop 6yr Est 15yr Est 9yr Mad 15yr Mad 9yr DK 15yr DK 9yr Figure 1: Odds ratios of observed number of children with 4 or 5 risk factors divided with expected number for each cohort. versus expected number of children. From these analyses clustering was found for ≥4 risk factors in all cohorts except the 6-year-old cohort from Copenhagen (CoSCIS) (Figure 1). The number of children having ≥3 risk factors also showed clustering, but with a low OR (web-only Figure A). 3.1. Sensitivity Analyses (Table 4). Analyses of composite MetS score were compared when sum of 4 skinfolds or BMI were used rather than waist circumference as the adiposity risk factor. Systolic BP (mmHg) M SD The Pearson correlation between the mean z-scores including either BMI, waist circumference, or skinfold, respectively, was >0.97 for all associations. When the associations between CRF and dichotomized variables of the three mean z-scores were examined, greater magnitude of association was found when sum of skinfolds was used as the adiposity variable in the composite score, but similar magni- tude was found for scores including waist circumference and BMI (Table 4). Sensitivity analysis was conducted to examine whether the magnitude of associations between CRF and the MetS score differed when HOMA was included in the score rather than fasting glucose, keeping other variables constant. The magnitude of association was greater when the MetS composite score included HOMA rather than fasting glucose (Table 4). Including APOA1, APOB, adiponectin, or CRP in the MetS score did not strengthen the association between CRF and MetS score even if they were associated with MetS score. However, leptin improved the composite MetS score substantially. OR for the least fit quartile increased from 18.3 (7.8–43.3) to 75.2 (18.2–309.8) when leptin was added to the M tS Th t th d i ti t Table 2: Overall mean value and regression 𝛽-values with risk factors and age. 𝛽-values were used to calculate age adjusted risk factor levels. Systolic BP (mmHg) M SD Journal of Diabetes Research 5 Table 2: Overall mean value and regression 𝛽-values with risk factors and age. 𝛽-values were used to calculate age adjusted risk factor levels. Characteristic Regression 𝛽for age Girls Boys Mean 𝛽 𝑃< Mean 𝛽 𝑃< BMI (kg/m2) 19.6 0.704 0.001 19.6 0.699 0.001 Waist circumference (cm) 67.4 2.190 0.001 68.3 2.317 0.001 Ratio waist to hip circumference 77.8 −0.014 0.001 75.6 0.012 0.001 Sum of 4 skinfolds (mm) 42.9 2.453 0.001 33.5 0.743 0.001 Diastolic BP (mmHg) 61.5 0.386 0.001 62.0 0.442 0.001 Systolic BP (mmHg) 105.2 1.339 0.001 106.1 1.382 0.001 Total cholesterol (mmol/L) 4.29 −0.032 0.001 4.10 −0.070 0.001 HDL-cholesterol (mmol/L) 1.48 −0.019 0.001 1.48 −0.025 0.001 LDL-cholesterol (mmol/L) 2.40 −0.039 0.001 2.29 −0.044 0.001 Triglyceride (mmol/L) 0.80 0.025 0.001 .75 0.028 0.001 Glucose (mmol/L) 4.99 0.041 0.001 5.06 0.042 0.001 Insulin (pmol/L) 55.0 4.476 0.001 51.1 4.522 0.001 CRF (VO2max [mL/min/kg]) 42.5 −0.634 0.001 50.5 0.418 0.001 CRP (mg/dL) .84 −0.015 NS .74 0.006 NS APOA1 (𝜇mol/L) 11.2 1.311 0.001 10.6 1.197 0.001 APOB (𝜇mol/L) 1.00 0.037 0.001 .94 0.032 0.001 Leptin (ng/mL) .93 0.099 0.001 .37 −0.007 NS Adiponectin (𝜇g/mL) 17.0 −0.396 0.001 15.2 −0.841 0.001 HOMA 12.6 1.111 0.001 11.8 1.129 0.001 Ln of OR for 4 + 5 RF 0.75 1 10 Combined Sw 12yr Sw 8yr N 15yr 2005 N 9yr 2005 US 14–18yr US 10–14yr Cop 13yr Cop 9yr Cop 6yr Est 15yr Est 9yr Mad 15yr Mad 9yr DK 15yr DK 9yr Figure 1: Odds ratios of observed number of children with 4 or 5 risk factors divided with expected number for each cohort. 3.1. Sensitivity Analyses (Table 4). Analyses of composite MetS score were compared when sum of 4 skinfolds or BMI were used rather than waist circumference as the adiposity risk factor. The Pearson correlation between the mean z-scores including either BMI, waist circumference, or skinfold, respectively, was >0.97 for all associations. When the associations between CRF and dichotomized variables of the three mean z-scores were examined, greater magnitude of association was found when sum of skinfolds was used as the adiposity variable in the composite score, but similar magni- tude was found for scores including waist circumference and BMI (Table 4). Systolic BP (mmHg) M SD However, leptin improved the composite MetS score substantially. OR for the least fit quartile increased from 18.3 (7.8–43.3) to 75.2 (18.2–309.8) when leptin was added to the MetS score. The strengthened association was apparent even if waist circumference was already part of the mean z-score. Ln of OR for 4 + 5 RF Figure 1: Odds ratios of observed number of children with 4 or 5 risk factors divided with expected number for each cohort. versus expected number of children. From these analyses clustering was found for ≥4 risk factors in all cohorts except the 6-year-old cohort from Copenhagen (CoSCIS) (Figure 1). The number of children having ≥3 risk factors also showed clustering, but with a low OR (web-only Figure A). Across cohorts, 6.2% of youth had 4+ risk factors and 15.7% had 3+ risk factors where a risk factor was defined as the upper quartile of risk (Table 3). OR for three risk factors was 1.09 (1.02–1.17) (online-only Figure 1B). The true number of children with clustering of CVD risk factors may be between 6.2% and 15.7%, and a conservative approach would be to define 6.2% with MetS. Cutoff in mean z-score for 6.2% was 0.85 and cutoff selecting 15.7% was 0.39. Further, the importance of including CRF as a component of the MetS composite score was tested. To do this we needed an independent variable to analyze against MetS. Waist was therefore removed from the MetS score and used as independent variable. In this analysis the MetS consisted of systolic BP, TG, inverse HDL, and HOMA. Additional 6 Journal of Diabetes Research 6 Table 3: Distribution of children according to the number of risk factors1. The two next columns display the proportion of children having 4 or more risk factors and the cutoff point in the continuous 𝑧-score2, which select the same number of children at risk. The last column displays proportion having MetS according to IDF definition3. Systolic BP (mmHg) M SD For each comparison a cutoff point in 𝑧-score was chosen, which selected the same proportion of cases Odds ratios (95% CI) for quartiles of fitness 1st quartile 2nd quartile 3rd quartile 4th quartile MetS without fatness (TG + HOMA + BP −HDL) 5.2 (3.8–7.2) 2.0 (1.4–2.9) 1.8 (1.2–2.6) 1 (ref.) MetS with waist 19.5 (10.8–35.1) 3.7 (2.0–7.1) 2.7 (1.4–5.3) 1 (ref.) MetS with sum4skin 30.0 (15.3–58.6) 5.0 (2.4–10.3) 3.2 (1.5–6.8) 1 (ref.) MetS with BMI 21.8 (12.2–39.3) 4.8 (2.5–9.0) 2.7 (1.4–5.2) 1 (ref.) MetS with bioimpedance (%fat)* 11.7 (5.2–26.6) 2.2 (0.8–5.6) 1.0 (0.3–3.0) 1 (ref.) MetS with waist circumference* 10.9 (4.2–28.4) 2.3 (0.8–6.9) 1.6 (0.5–5.2) 1 (ref.) MetS with HOMA 19.5 (10.8–35.1) 3.7 (2.0–7.1) 2.7 (1.4–5.3) 1 (ref.) MetS with glucose 13.0 (7.7–21.8) 2.3 (1.3–4.3) 1.2 (0.6–2.4) 1 (ref.) MetS (TG + HOMA + sysBP + waist −HDL) 18.3 (7.8–43.3) 3.2 (1.2–8.6) 2.6 (1.0–7.1) 1 (ref.) Adding leptin 75.2 (18.2–309.8) 7.6 (1.6–35.4) 5.2 (1.1–25.0) 1 (ref.) MetS (TG + HOMA + sysBP + waist −HDL) 22.7 (10.8–47.7) 3.1 (1.3–7.5) 2.3 (0.9–5.6) 1 (ref.) Adding APOA1 19.8 (9.8–40.1) 2.8 (1.2–6.4) 2.0 (0.8–4.8) 1 (ref.) Adding APOB 24.3 (10.4–56.7) 4.5 (1.7–11.6) 2.5 (0.9–7.0) 1 (ref.) Adding adiponectin 13.5 (6.5–27.9) 1.9 (0.8–4.9) 2.0 (0.8–4.7) 1 (ref.) Adding CRP 12.7 (7.4–21.7) 2.8 (1.5–5.3) 1.8 (1.0–3.5) 1 (ref.) MetS (TG + HOMA + BP −HDL) against CRF quartiles# 5.2 (3.8–7.2) 2.0 (1.4–2.9) 1.8 (1.2–2.6) 1 (ref.) MetS (TG + HOMA + BP −HDL) against waist quartiles# 1 (ref.) 1.1 (0.8–1.6) 1.2 (0.9–1.7) 4.1 (3.1–5.4) Adding CRF against waist quartiles 1 (ref.) 1.8 (1.1–3.0) 2.5 (1.5–4.1) 14.8 (9.6–22.9) #These two analyses compare the strength between quartiles of fitness and quartiles of waist circumference against the same MetS outcome. *Data only available in CoSCIS, which is why estimates of quartiles of fitness in relation to MetS including waist circumference differ from analysis above. the number fulfilling IDF definition of MetS for children. More than 6.2% of children had clustering of 4 or more CVD risk factors in contrast to less than 1% according to the IDF definition. Changing the IDF diagnostic criteria to include more children would enable health professionals to start preventive initiatives much earlier and using a noninvasive screening tool may allow school teachers to start counseling health behavior in children at risk. Systolic BP (mmHg) M SD If IDF definition only diagnoses about one-tenth of children with metabolic risk, a change in definition is necessary, because early prevention may be much more efficient than later treatment of manifest disease. inclusion of CRF increased OR for the highest quartile of waist circumference from around 4 to 15, compared to the lowest quartile. We also analyzed waist circumference and CRF, indepen- dently, as exposure variables against a MetS score including systolic BP, HOMA, TG, and inverse HDL. A higher OR was found for low CRF compared to high waist circumference. In the analysis of waist circumference, the 2nd and 3rd quartile of waist were not different from the lowest quartile and the upper quartile had an increased risk of 4.1 (95% CI 3.1–5.4) (Table 4). For CRF all three lower quartiles had higher risk than the most fit with an OR of 5.2 (3.8–7.2) for the least fit. Previous definitions have used dichotomized risk factors to define MetS. The use of cutoff points in CVD risk factors could originate in previous use of guidelines to determine drug treatment, but drug treatment may not be the first choice in children. A continuous score includes full information of the health status from the risk factors. Continuous scores have recently been used by several groups [22, 23]. Some authors have postulated that z-scores are population specific [24]. However, this is only true if mean and SD from the specific population are used when variables are standardized. We have used common mean and SD of age and sex adjusted variables calculated from a great number of cohorts, which strengthen the generalizability of the score. We suggest changing existing dichotomized definitions to use continuous scores, because these use full information and can be easily calculated using Internet-based equations. 3.2. Testing a Noninvasive MetS Score. The noninvasive MetS composite score (mean of z-score for waist circumference/ height and 1/CRF) was analyzed against the IDF definition of MetS using ROC analysis. A sensitivity and specificity of 0.85 and an area under the curve of 0.92 were found for a threshold point of 0.51 in z-score. When analyzed against the mean z- score of log waist, log TG, log HOMA, systolic BP, inverse HDL, and inverse fitness, a noninvasive mean z-score of 0.44 gave a sensitivity and specificity of 0.87 with an area under the curve of 0.94. Systolic BP (mmHg) M SD Cohort Number with the risk factors waist, sysBP, 1/HDL, TG, and HOMA Total 𝑁Proportion (%) Mean 𝑧-score2 Proportion (%) using IDF definition3 0 RF 1 RF 2 RF 3 RF 4 RF 5 RF 4 + 5 RF Denmark 9 yr (EYHS) 302 306 191 85 36 8 928 4.74 0.83 0.0 Denmark 15 yr (EYHS) 260 261 200 67 36 7 831 5.17 0.88 0.1 Portugal 9 yr (EYHS) 374 271 171 105 52 22 995 7.44 0.63 0.5 Portugal 15 yr (EYHS) 231 240 150 66 30 8 725 5.24 0.46 0.2 Estonia 9 yr (EYHS) 178 177 110 64 26 5 560 5.54 0.76 0.0 Estonia 15 yr (EYHS) 184 190 122 66 21 9 592 5.07 0.82 0.2 Denmark 6 yr (CoSCIS) 153 137 118 48 9 2 467 2.36 0.80 0.0 Denmark 9 yr (CoSCIS) 160 132 80 37 15 8 432 5.32 0.91 0.2 Denmark 13 yr (CoSCIS) 156 132 83 41 23 10 445 7.42 0.81 0.0 USA 10–14 yr (NHANES) 158 132 53 43 28 12 426 9.39 1.36 3.0 USA 14–18 yr (NHANES) 351 241 124 83 49 22 870 8.42 1.29 4.1 Norway 9 yr (EYHS 2005) 352 289 191 92 48 21 893 6.95 0.72 0.8 Norway 15 yr (EYHS 2005) 242 241 148 67 33 11 742 5.93 0.80 0.9 Switzerland 8 yr (KISS) 132 118 73 28 16 9 376 6.70 0.65 0.3 Switzerland 12 yr (KISS) 181 128 95 55 22 8 489 2.30 0.90 0.0 Total 3212 2793 1794 900 405 149 9871 6.20 0.85 0.9 1At risk factor is defined as the upper quartile. 2Mean 𝑧-score was calculated after ln-transformation of skewed risk factors. 3 f h ld h S d fi d ff d b Z l ( ) [ ] 7 Journal of Diabetes Research Table 4: Sensitivity analyses of changing one parameter in the MetS outcome with another covering the same trait. At the bottom CRF and waist circumference are compared against the same MetS outcome. For each comparison a cutoff point in 𝑧-score was chosen, which selected the same proportion of cases. Table 4: Sensitivity analyses of changing one parameter in the MetS outcome with another covering the same trait. At the bottom CRF and waist circumference are compared against the same MetS outcome. 4. Discussion Clustering of CVD risk factors could be viewed as a biological sign of poor metabolic health. The current analysis showed that more children had clustering of CVD risk factors than Journal of Diabetes Research 8 systolic BP, TG, HOMA, and inverse of HDL, we found an even stronger association than was found for quartiles of waist circumference analysed against the same outcome. CRF may be the most important risk factor, which should have been included in definitions of MetS, but leptin also strengthened the association between the MetS variable and the independent variable. There is consensus that insulin resistance is a key factor in clustering of CVD risk factors and that muscle tissue is quantitatively the largest organ for glucose disposal. It has been shown that one-legged training can double glucose uptake in the trained leg compared to the untrained, which proves the importance of muscle tissue independent of abdominal obesity in the development of insulin resistance [30]. The methodology to diagnose MetS should include existing accepted risk factors but should allow for other emerging risk factors, which may prove important in future research. Inclusion of CRF and leptin improved the MetS score. Because we use the mean of standardized variables, scores are still comparable between studies. Calculations may seem cumbersome, but we have constructed a freely available software on the Internet to calculate scores, which makes it easy to use in general practise (http://www.video4coach.com/zscore/index.html). Sensitivity analyses showed that CRF was as strongly related to other CVD risk factors as waist circumference and logically should be included in MetS definition. Includ- ing leptin strengthened the association between MetS and an exposure substantially even if waist circumference was already included in the score. Inclusion of CRP, adiponectin, APOA1, and APOB did not strengthen the association fur- ther. A general practitioner is not expected to perform the CRF test, but all school physical education teachers can perform field tests of CRF with sufficient quality. A number of field tests are available, which allow whole school classes to be tested in one session [25, 26]. The IDF definition also uses fasting glucose as criteria for an adverse glucose homeostasis, which makes sense in adults where 𝛽-cell function has decreased. However, HOMA score provides a better estimate of impending or current impaired glucose metabolism in children. This view is supported by other studies [27]. Disclaimer The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The study is not submitted elsewhere. All authors had full access to the data and can take responsibility for the integrity of the data and the accuracy of the data analyses. f Inclusion of a large number of cohorts sampled from North-, East-, Mid-, and Southern Europe and North America enables comparison of consistency of observations between populations, which is a strength to this study. In these culturally diverse populations the clustering of risk factors occurred at a similar level of mean z-score. The approach is flexible in relation to which risk factors, covering a specific trait, are available. 4. Discussion Inclu- sion of other risk factors than the traditional IDF factors in calculation of mean z-score is easy and strengthens diagnostic criteria, which improve sensitivity and specificity. However, even if some risk factors are not measured, the mean z- score may still indicate a reasonable correct level of metabolic health.i A weakness of the IDF definition is the use of 90% percentile of waist circumference to define overweight [6]. This definition makes it difficult to compare populations, and it also hides secular trend in a population where a preventive strategy could be justified, because WC have gradually changed over time. Sensitivity analysis showed that fatness variables were interchangeable. This means that the use of continuous, age adjusted, and standardized variables in the definition of MetS makes it possible to compare risk levels between populations and over time even if studies have used different variables. However, we still recommend using waist circumference or BMI, because skinfold measurement is difficult to standardize. Variables included in the z-score should be constructed from a fixed mean and SD. In conclusion, the use of CVD risk factors as continuous variables can provide more information on MetS in children. Also, inclusion of other risk factors such as CRF and leptin and substituting glucose with HOMA score improved diag- nostic criteria. This is important since most children do not have CVD but are in the developmental stages of MetS. The suggested approach solves many of the inherent problems in the previous definition formulated by IDF. Further, our noninvasive approach may be useful as a prescreening tool. Disclosure Lars Bo Andersen affirms that the paper is an honest, accurate, and transparent account of the study being reported and that no important aspects of the study have been omitted. 4.1. Strengths and Weaknesses. Assessment of the prognostic value of MetS in children for later CVD constitutes a weak- ness. The composite MetS score tracks into adulthood [28], but it is still uncertain how risk level in a child is related to later CVD. 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https://openalex.org/W2894749230	https://europepmc.org/articles/pmc6331457?pdf=render	English	null	Endogenous retroviruses transcriptional modulation after severe infection, trauma and burn	bioRxiv (Cold Spring Harbor Laboratory)	2,018	cc-by	10,476	Endogenous Retroviruses Transcriptional Modulation After Severe Infection, Trauma and Burn Olivier Tabone 1, Marine Mommert 1,2, Camille Jourdan 1, Elisabeth Cerrato 1, Matthieu Legrand 3, Alain Lepape 4, Bernard Allaouchiche 4,5, Thomas Rimmelé 1, Alexandre Pachot 1, Guillaume Monneret 1,6, Fabienne Venet 1,6, François Mallet 1,2 and Julien Textoris 1,7* Olivier Tabone 1, Marine Mommert 1,2, Camille Jourdan 1, Elisabeth Cerrato 1, Matthieu Legrand 3, Alain Lepape 4, Bernard Allaouchiche 4,5, Thomas Rimmelé 1, Alexandre Pachot 1, Guillaume Monneret 1,6, Fabienne Venet 1,6, François Mallet 1,2 and Julien Textoris 1,7* 1 EA7426 Hospices Civils de Lyon–bioMérieux–UCBL1 “Pathophysiology of Injury Induced Immunosuppression,” Groupement Hospitalier Edouard Herriot, Lyon, France, 2 Joint Research Unit, Hospice Civils de Lyon, bioMerieux, Centre Hospitalier Lyon Sud, Pierre-Benite, France, 3 Department of Anesthesiology and Critical Care and Burn Unit, Groupe Hospitalier St-Louis-Lariboisière, Assistance publique-Hôpitaux de Paris, Paris, France, 4 Hospices Civils de Lyon, Intensive Care Unit, Centre Hospitalier Lyon Sud, Pierre Bénite, France, 5 Agressions Pulmonaires et Circulatoires dans le Sepsis APCSe VetAgro Sup UPSP 2016.A101, Centre Hospitalier Lyon-Sud, Pierre Bénite, France, 6 Hospices Civils de Lyon, Immunology Laboratory, Groupement Hospitalier Edouard Herriot, Lyon, France, 7 Hospices Civils de Lyon, Department of Anaesthesiology and Critical Care Medicine, Groupement Hospitalier Edouard Herriot, Université Claude Bernard Lyon 1, Lyon, France Keywords: endogenous retroviruses, severe inﬂammatory injuries, septic shock, burn, trauma, transcriptome, host response Correspondence: Julien Textoris julien.textoris@biomerieux.com Specialty section: This article was submitted to Viral Immunology, a section of the journal Frontiers in Immunology Specialty section: This article was submitted to Viral Immunology, a section of the journal Frontiers in Immunology Received: 11 July 2018 Accepted: 13 December 2018 Published: 08 January 2019 Received: 11 July 2018 Accepted: 13 December 2018 Published: 08 January 2019 Edited by: Shokrollah Elahi, University of Alberta, Canada Reviewed by: Alexander Emmer, Martin Luther University of Halle-Wittenberg, Germany Giuseppe Bertoni, Institute of Virology and Immunology (IVI), Switzerland Although human endogenous retroviruses (HERVs) expression is a growing subject of interest, no study focused before on speciﬁc endogenous retroviruses loci activation in severely injured patients. Yet, HERV reactivation is observed in immunity compromised settings like some cancers and auto-immune diseases. Our objective was to assess the transcriptional modulation of HERVs in burn, trauma and septic shock patients. We analyzed HERV transcriptome with microarray data from whole blood samples of a burn cohort (n = 30), a trauma cohort (n = 105) and 2 septic shock cohorts (n = 28, n = 51), and healthy volunteers (HV, n = 60). We described expression of the 337 probesets targeting HERV from U133 plus 2.0 microarray in each dataset and then we compared HERVs transcriptional modulation of patients compared to healthy volunteers. Although all 4 cohorts contained critically ill patients, the majority of the 337 HERVs was not expressed (around 74% in mean). Each cohort had differentially expressed probesets in patients compared to HV (from 19 to 46). Strikingly, 5 HERVs were in common in all types of severely injured patients, with 4 being up-modulated in patients. We highlighted co-expressed proﬁles between HERV and nearby CD55 and CD300LF genes as well as autonomous HERV expression. We suggest an inﬂammatory-speciﬁc HERV transcriptional response, and importantly, we introduce that the HERVs close to immunity-related genes might have a role on its expression. ORIGINAL RESEARCH published: 08 January 2019 doi: 10.3389/ﬁmmu.2018.03091 ORIGINAL RESEARCH published: 08 January 2019 doi: 10.3389/ﬁmmu.2018.03091 ORIGINAL RESEARCH published: 08 January 2019 doi: 10.3389/ﬁmmu.2018.03091 Abbreviations: HERV, Human endogenous retrovirus; LTR, Long Terminal Repeats; PAMP, pathogen-associated molecular pattern; LPS, Lipopolysaccharide; PMA, phorbol-12-myristate-13-acetate; HV, healthy volunteers; ICU, Intensive Care Unit; TBSA, Total Burn Surface Area; ABSI, Abbreviated Burn Severity Index; ISS, Injury Severity Score; SAPSII, Simpliﬁed Acute Physiology Score II; MFI, Medians of Fluorescence Intensity. Citation: Tabone O, Mommert M, Jourdan C, Cerrato E, Legrand M, Lepape A, Allaouchiche B, Rimmelé T, Pachot A, Monneret G, Venet F, Mallet F and Textoris J (2019) Endogenous Retroviruses Transcriptional Modulation After Severe Infection, Trauma and Burn. Front. Immunol. 9:3091. doi: 10.3389/ﬁmmu.2018.03091 January 2019 \| Volume 9 \| Article 3091 Frontiers in Immunology \| www.frontiersin.org 1 HERV Modulation in Acute Inﬂammatory Diseases Tabone et al. Traumas cohort 105 i i 105 patients with severe trauma were admitted at HCL. Brieﬂy, patients were included when they were under mechanic ventilation, with an Injury Severity Score (ISS) over 25 and were at least 18 years old. Inclusion / exclusion criteria and ethical considerations of the cohort have been previously published elsewhere (24). The main clinical variables are summarized on Table S1. Samples were collected at day 1 (D1) or day 2 (D2) after trauma. Data from 22 healthy volunteers were also used to make comparisons with patients (identical with septic shock cohort 2). Few studies focused on HERVs reactivation in acute inﬂammatory contexts. In mice, modulation of HERVs expression has been shown to be quite speciﬁc, with signatures related to pathogen-associated molecular pattern (PAMPs) (12). In human, LPS or PMA stimulations of myeloid cells revealed an increase expression of four HERVs families (13). In vivo, HERVs expression has been detected in the plasma and whole blood samples of burn patients (14, 15) although the studies focused on whole HERVs families, not on speciﬁc loci. Studying HERV transcriptome modulation after severe inﬂammatory injuries could help to better understand pathological states of patients. INTRODUCTION of commercial whole genome microarray do target HERV loci (21, 22) (such as Aﬀymetrix U133 plus 2), we retrospectively explored microarray datasets obtained in our lab to study the HERV transcriptome modulation in various contexts of injuries in vivo. Human Endogenous Retroviruses (HERVs) are former exogenous retroviruses which have infected germinal cells and became integrated in our genome million years ago (1). These rare events happened several times in evolution. As retrotransposons, they are able to duplicate across the genome and they represent today more than 8% of our genome. Each insertion therefore led to distinct groups or families, each including multiple copies. Current classiﬁcation annotates around 100 such groups. Burns cohort 30 severe burn patients admitted at Hospices Civils de Lyon, France (HCL) were included in a placebo-controlled, randomized, double-blind study assessing the eﬃcacy of hydrocortisone administration on burn shock duration. Inclusion / exclusion criteria, clinical description and ethical considerations of the cohort have been previously published elsewhere (16, 23). Thirteen healthy volunteers were also recruited within Hospices Civils de Lyon to serve as controls for the transcriptional study. Whole blood samples were collected at inclusion (severe shock, before any treatment, Day 1) and in the following days (around day 2 (D2), day 5 (D5), and day 7 (D7) after inclusion). HERV loci initially shared a common structure with exogenous retroviruses: internal protein coding regions (gag, pro, pol, env) ﬂanked by two identical Long Terminal Repeats (LTRs). The accumulation of mutations and recombination events during evolution made most of these elements incomplete and defective for replication. Indeed, most of HERVs in our genome are now solo LTRs (1) resulting from recombination between 5 ′ and 3 ′ proviral LTRs. LTRs are critical elements that control viral gene expression either as promoters, enhancers or as polyadenylation signals. When inserted upstream, within or downstream of a “conventional” protein coding gene, LTRs can modulate its expression pattern (2, 3). For example, the presence of intronic LTR can result in novel transcripts, by providing alternative promoters, enhancers or polyadenylation signals, or by altering RNA splicing (4–6). Very few is known about of the transcriptional modulation of such elements in pathological contexts but in cancers [like testicular cancer (7) or colorectal cancer (8)] and auto-immune diseases [like multiple sclerosis (9–11)]. Septic shock cohort 1(SS1) Twenty-eight septic shock patients and 25 HV admitted into 2 ICUs of HCL were included in this study to explore the early transcriptome modulation after septic shock. Inclusion/exclusion criteria, clinical description and ethical considerations of the cohort have been previously published elsewhere (25). The ﬁrst blood sample was collected at the onset of shock (i.e., within 30 min after the beginning of vasoactive treatment, D0) and at day 1 (D1) and day 2 (D2) after shock. After severe injuries like septic shock, burn or trauma, leading to an important inﬂammatory response, we and others have shown that the blood transcriptome is highly modulated, with early and profound changes in adaptive and innate immune responses (16, 17). Moreover, in these contexts, viral reactivation is often observed, especially for Herpes Viruses (18, 19). This reactivation is associated with an immunosuppressive state (20). We therefore hypothesize that HERV, like latent viruses, may reactivate and be transcribed in vivo after inﬂammatory injuries. Given that several groups showed that some probes Septic shock cohort 2 (SS2) Fifty-one septic shock patients admitted to two Intensive Care Units (ICU) of HCL and 22 HV were included in a prognostic biomarker study. Inclusion / exclusion criteria, clinical description and ethical considerations of the cohort have been previously published elsewhere (26). Samples were collected at day 1 (D1), day 2 (D2) and day 3 (D3) after shock. Reverse Transcription and Quantitative PCR Whole blood samples (EDTA tubes) from 22 septic shock patients sampled at D1/D2, D3/D4/D5, and D6/D7/D8 after shock and admitted in Edouard Herriot hospital at Lyon, France were recruited as part of IMMUNOSEPSIS study (NCT02803346). Patients were aged from 23 to 81 (mean = 68), 16 men. Eleven samples were used for CD300LF protein analysis and 11 other samples for CD55 protein analysis. RNA from the cohorts, according to the above criteria, and new RNA from HV were selected. RNA concentration was determined using Quant-iT RNA, BR assay on Qubit (Life Technologies, Chicago, Ilinois, United States). RNA integrity was assessed with the RNA 6000 Nano Kit on a Bioanalyzer (Agilent Technologies, Santa Clara, California, United States). Samples with RNA integrity number ≤6 were excluded due to poor quality RNA. Total RNA was reverse transcribed in complementary DNA (200 ng in a ﬁnal volume of 20 µL) using QuantiTect Reverse Transcription kit (Qiagen) as recommended by the manufacturer. The expression levels of genes (CD55, CD300LF, SLC8A1, NFE4, PTTG1IP, and HPRT1 as reference gene) and associated HERVs were quantiﬁed using quantitative-real time polymerase chain reaction (qPCR). qPCR were performed on a LightCycler instrument using Light Cycler 480 Probes Master for the genes and reference genes and on SYBR Green I master for HERVs. Final volume of 20 µL contains 0.5 µM of primers. For genes, an initial denaturation step of 10 min at 95◦C followed by 45 cycles, 10 s at 95◦C, 29 s annealing at 60◦C, and 1 s extension at 72◦C, Taqman) was performed. For HERVs, an initial denaturation step of 5 min at 95◦C followed by 45 cycles of a PCR protocol (10 s at 95◦C, 15 s at 55◦C and 15 s at 72◦C, SYBR Green program), melting curve protocol was performed. The Second Derivative Maximum Method was used with the LightCycler software (Release 1.5.1) to automatically determine the crossing point for individual samples. Standard curves were generated by using serial dilutions of cDNA standards prepared from puriﬁed PCR amplicons obtained with the corresponding primers (Table S2). Relative standard curves describing the PCR eﬃciency of selected targets were created and used to perform Microarray Analysis Microarray data are available on the Gene Expression Omnibus (GEO) website for Burn [GEO:GSE77791], SS1 [GEO:GSE57065], and SS2 [GEO:GSE95233] cohorts. The preprocessing methods were comparable in all datasets. Microarray normalization and statistical analysis were performed using R/Bioconductor (R v3.2.3). Quality assessment was performed through simpleaﬀy (v2.46.0) (27). After removing outlier samples the raw data were normalized, adjusted for background noise and summarized using the GCRMA (Guanine Cytosine Robust Multi-Array) algorithm with default parameters (28). COMBAT algorithm (29) was used to remove batch eﬀect on Burn and Trauma cohorts. The 337 probesets from the U133 Plus2.0 microarray targeting HERVs have been identiﬁed and selected as described elsewhere (21, 22). Healthy volunteers Whole blood samples (EDTA tubes) were purchased from the Etablissement Français du Sang (n = 18). Donors were aged from 21 to 63 (mean = 50), 12 men and 6 women. They were age-matched with burn and septic shock cohorts. According to the standardized procedure for blood donation, written informed consent was obtained from healthy volunteers (HVs) and personal data for blood donors were anonymized at time of blood donation and before blood transfer to a research lab. Burns Whole blood samples (EDTA tubes) from 13 burn patients sampled at D1 and D7 and admitted in Edouard Herriot hospital at Lyon, France were recruited as part of the EARLYBURN study (NCT02940171). Patients were aged from 21 to 84 (mean = 53), 12 men. The mean TBSA was 33% (from 20 to 52%). All samples from these patients were used for CD300LF protein analysis, and 7 of these 13 patients were used for CD55 protein analysis. All the analysis were made with R (3.2.3). The diﬀerential expression analysis was performed with Limma package (3.26.9) (30). A probeset was considered signiﬁcantly statistically diﬀerentially expressed between two conditions when absolute log2 Fold Change was higher than 0.5 and adjusted P-values [Benjamini-Hochberg correction (31)] lower than 0.01. Patients Subset of cohorts used for microarray analysis were used for validation cohort: 10 burn samples at D1, 10 traumas samples at D1, 10 SS1 samples at D1, 10 SS2 samples at D1. Each subset was January 2019 \| Volume 9 \| Article 3091 Frontiers in Immunology \| www.frontiersin.org 2 HERV Modulation in Acute Inﬂammatory Diseases Tabone et al. (Aﬀymetrix) and images (DAT ﬁles) were converted to CEL ﬁles using GCOS software (Aﬀymetrix). matched with its corresponding cohort on: Age, sex and Total Burn Surface Area (TBSA) for burns-Sex, Sepsis at D7 and Death at D28 for traumas-Age, sex and SAPS II for SS1-Age, Sex and Death at D28 for SS2. Healthy Volunteers Whole blood samples were purchased from the Etablissement Français du Sang (n = 12). The mean age of HV is 56, with a standard error of 9. According to the standardized procedure for blood donation, written informed consent was obtained from healthy volunteers (HVs) and personal data for blood donors were anonymized at time of blood donation and before blood transfer to a research lab. January 2019 \| Volume 9 \| Article 3091 Statistics Wilcoxon signed rank tests were done for RT-qPCR and ﬂow cytometry results, by comparison between HV and each cohort of patients, for each target. ETHICS APPROVAL AND CONSENT TO PARTICIPATE In order to analyze the HERV transcriptome modulation associated with injury, we performed a supervised analysis comparing HERV expression in injured patients at D1 (admission) and HV, in each dataset separately. The comparison (accounting for multiple testing correction with absolute fold change higher than 1 and corrected p-value lower than 0.01) between burn patients and HV resulted in 19 diﬀerentially expressed HERVs (Supplemental Figure 3A). The comparison between trauma patients and HV resulted in 27 diﬀerentially expressed HERVs (Supplemental Figure 3B). The comparison between septic shock patients and HV resulted in 19 and 46 diﬀerentially expressed HERVs for cohorts 1 and 2 respectively (Supplemental Figures 3C,D). Altogether, 56 distinct probesets targeting HERVs were diﬀerentially expressed among all 4 datasets, clearly discriminating HV from patients at EDTA blood tubes were obtained from EFS (Etablissement Français du Sang) and used immediately. In accordance with EFS standardized procedures for blood donation, written no- objection was obtained from healthy volunteers to use the blood for the research and personal data for blood donors were anonymized before blood transfer to our research lab. Protocols of the discovery and validation cohorts were approved by local ethics committees. Non-opposition to inclusion in the protocols was systematically recorded from patients or next of kin. Flow Cytometry Sampling and Staining The following antibodies were used: anti CD14-BV510, anti CD3–BV421 and anti CD56–PECy7 from BD Biosciences; anti CD300lf-PE from BD Biosciences or anti CD55-APC from Biolegend; anti CD16-APC from BD Biosciences or anti CD16- FITC from Beckman Coulter (Miami, FL) and PE Mouse IgG1, κ Isotype Control from BD Biosciences or APC Mouse IgG1, κ Isotype Control from R&D System. Red blood cell lysis was performed using Versalyse lysing solution (Beckman Coulter). CD300LF and CD55 expression were measured using Navios ﬂow cytometer (Beckman-Coulter). Results were analyzed with Kaluza software (Beckman-Coulter) expressed as Medians of Fluorescence Intensity (MFI). ETHICS APPROVAL AND CONSENT TO PARTICIPATE genome U133 plus 2.0 microarray datasets. Among them, a majority had low expression levels, within background levels (Supplemental Figure 1). Based on hierarchical clustering analysis, 64 probesets (19%) were expressed (i.e., above background) for burns, 60 probesets (18%) for traumas, 164 for septic shock 1 (49%) and 63 for septic shocks 2 (19%). The 25% most variant probesets (n = 84) across samples in each dataset revealed that several probesets were even highly expressed (Figure 1). In each dataset, the hierarchical clustering highlighted a clear diﬀerence between patients and HV, suggesting a modulation of HERV expression following injury. Interestingly, over these top 25% most variant probesets selected in each dataset (resulting of 127 distinct probesets), 44 (35%) were similarly modulated in the four datasets, and 102 (80%) in at least 2 datasets (Supplemental Figure 2). In order to analyze the HERV transcriptome modulation associated with injury, we performed a supervised analysis comparing HERV expression in injured patients at D1 (admission) and HV, in each dataset separately. The comparison (accounting for multiple testing correction with absolute fold change higher than 1 and corrected p-value lower than 0.01) between burn patients and HV resulted in 19 diﬀerentially expressed HERVs (Supplemental Figure 3A). The comparison between trauma patients and HV resulted in 27 diﬀerentially expressed HERVs (Supplemental Figure 3B). The comparison between septic shock patients and HV resulted in 19 and 46 diﬀerentially expressed HERVs for cohorts 1 and 2 respectively (Supplemental Figures 3C,D). Altogether, 56 distinct probesets targeting HERVs were diﬀerentially expressed among all 4 datasets, clearly discriminating HV from patients at genome U133 plus 2.0 microarray datasets. Among them, a majority had low expression levels, within background levels (Supplemental Figure 1). Based on hierarchical clustering analysis, 64 probesets (19%) were expressed (i.e., above background) for burns, 60 probesets (18%) for traumas, 164 for septic shock 1 (49%) and 63 for septic shocks 2 (19%). The 25% most variant probesets (n = 84) across samples in each dataset revealed that several probesets were even highly expressed (Figure 1). In each dataset, the hierarchical clustering highlighted a clear diﬀerence between patients and HV, suggesting a modulation of HERV expression following injury. Interestingly, over these top 25% most variant probesets selected in each dataset (resulting of 127 distinct probesets), 44 (35%) were similarly modulated in the four datasets, and 102 (80%) in at least 2 datasets (Supplemental Figure 2). Frontiers in Immunology \| www.frontiersin.org RNA Extraction and Microarrays y Total RNA was extracted with PAXgeneTM Blood RNA kit (PreAnalytix, Hilden, Germany). Whole blood from PAXGeneTM tubes was preferred to either buﬀy coat or PBMCs to ensure reproducibility and avoid missing samples within the context of a clinical study. RNA integrity was assessed using Agilent 2100 Bioanalyser (Agilent Technologies, Waldbrom, Germany) and Lab-on-chip RNA 6000 Nano Assay (Agilent Technologies). Double-stranded cDNA was prepared from total RNA and an oligo-dT primer using GeneChip One-Cycle cDNA Synthesis Kit (Aﬀymetrix, Santa Clara, United States). Three microgram labeled cRNA were hybridized onto Human Genome U133 Plus 2.0 GeneChips (Aﬀymetrix), revealed and washed using FS450 ﬂuidic station. GeneChips were scanned using a 5G scanner January 2019 \| Volume 9 \| Article 3091 Frontiers in Immunology \| www.frontiersin.org 3 HERV Modulation in Acute Inﬂammatory Diseases Tabone et al. TABLE 1 \| Patients characteristics of burn, trauma, and septic shock cohorts included in microarray analyses. Variable Burn Trauma Septic shock 1 Septic shock 2 (n = 30) (n = 105) (n = 28) (n = 51) Age, years 48 (39–55) 38 (25–54) 62 (54–76) 65 (53–74) Gender, women, n (%) 8 (27%) 34 (32%) 9 (32%) 18 (35%) Weight, kg 94 (77–104) 78 (67–92) – – TBSA (%) 70 (48–84) – – – Baux score 110 (102–125) – – – ABSI score 11 (10–12) – – – ISS score – 34 (29–41) – – SOFA score – 5 (1–7) 10 (9–13) 10 (8–12) SAPSII score – 44 (29–56) 45 (34–56) 51 (43–62) Secondary septic shock 12 (40%) 29 (28%) – – ICU length of stay, days 66 (22–89) 9 (5–17) 10 (5–14) – D28 mortality, n (%) 8 (27%) 4 (4%) 5 (18%) 17 (33%) TBSA, Total Burn Surface Area (severe patient > 30%); Baux score, Predictor of mortality due to severe burns (severe patient > 100); ABSI, Abbreviated Burn Severity Index (severe patient > 9); ISS, Injury Severity Score (severe patient > 15); SOFA, Sequential organ failure assessment score (severe patient ≥3); SAPSII, Simpliﬁed Acute Physiology Score II. (severe patient > 30). In each cohort, cumulative mortality has been assessed up to the 28th day after ICU admission. TABLE 1 \| Patients characteristics of burn, trauma, and septic shock cohorts included in microarray analyses. eﬃciency-corrected quantiﬁcation with the LightCycler Relative Quantiﬁcation Software. Targets expression normalization was performed using a selected housekeeping gene (hypoxanthine phosphoribosyltransferase 1 [HPRT1, (32)]), and results were expressed as normalized concentration ratio. RESULTS (A) Expression levels in burn patients. (B) Expression levels in trauma patients. (C) Expression levels in septic shock 1 patients. (D) Expression levels in septic shock 2 patients. ICU admission (Supplemental Figure 4 and Table S3). Taking into account the global proﬁle for each probeset, 16 (28.6%) had higher expression in patients compared to HV and 40 (71.4%) were down-modulated in patients. Interestingly, 5 probesets were diﬀerentially expressed in all 4 datasets and 16 in at least 3 of them (Figure 2A). All 5 commonly modulated probesets had consistent expression proﬁle across the 4 datasets. Four were over-expressed in patients compared to healthy volunteers (Figure 2B). The 5th probeset, down-modulated in all datasets, maps at multiple locations in the genome and was not considered in further analyses. Among the 4 remaining modulated probesets, 1 HERV from ERV24B_Prim-int family (236982_at), is within 2kb from the PTTG1IP gene and 3 are within a gene. A HERV from LTR33 family (230354_at) is within an intron of SLC8A1 gene. A HERV from MLT1H family (1556107_at) and one from LTR16B2 family (1559777_at) are located in the 3’UTR of CD55 and MIR3945HG genes respectively (Table 2). to HV, decreasing over the ﬁrst week toward HV expression level at D7 (Supplemental Figure 5). The second one targets a LTR101_Mam HERV located in a 3 ′UTR of a processed transcripts of NFE4 gene. It was diﬀerentially expressed in the 2 septic shock cohorts. This probeset had the highest log2FC among the 5 septic shock-speciﬁc modulated probesets. ICU admission (Supplemental Figure 4 and Table S3). Taking into account the global proﬁle for each probeset, 16 (28.6%) had higher expression in patients compared to HV and 40 (71.4%) were down-modulated in patients. Interestingly, 5 probesets were diﬀerentially expressed in all 4 datasets and 16 in at least 3 of them (Figure 2A). All 5 commonly modulated probesets had consistent expression proﬁle across the 4 datasets. Four were over-expressed in patients compared to healthy volunteers (Figure 2B). The 5th probeset, down-modulated in all datasets, maps at multiple locations in the genome and was not considered in further analyses. Among the 4 remaining modulated probesets, 1 HERV from ERV24B_Prim-int family (236982_at), is within 2kb from the PTTG1IP gene and 3 are within a gene. A HERV from LTR33 family (230354_at) is within an intron of SLC8A1 gene. RESULTS We studied the in vivo modulation of the HERV transcriptome in three clinical relevant models of acute inﬂammatory injury: a burn, a trauma and 2 septic shock cohorts. We analyzed expression from each cohort independently comparing patients with healthy volunteers. All cohorts included severely injured patients (Table 1). The 30 burn patients had a median total burn surface area (TBSA) of 70% and high severity scores [median Baux: 110, median Abbreviated Burn Severity Index (ABSI): 11]. The 105 trauma patients had a median Injury Severity Score (ISS) score of 34 and a median Simpliﬁed Acute Physiology Score II (SAPSII) of 44. The 28 septic shocks from SS1 cohort had a median SAPSII of 45 and a median Charlson score of 2. The 51 patients from SS2 cohort had a median SAPSII of 51. As previously published (21, 22), we extracted data from 337 probesets targeting HERVs loci from the whole January 2019 \| Volume 9 \| Article 3091 Frontiers in Immunology \| www.frontiersin.org 4 HERV Modulation in Acute Inﬂammatory Diseases Tabone et al. FIGURE 1 \| Heatmap representation of HERVs in three models of injury. Heatmap of the 25% most variant probesets targeting HERVs in the four datasets: burn, trauma, and 2 septic shock cohorts. Probesets are in rows and samples in columns. Samples are annotated (colored bars on the top) by type of samples (HV in pink, patients in cyan) and day after inclusion (blue scaled). Expression levels are color-coded from blue (low expression) to red (high expression). Similar patterns of expression are highlighted through hierarchical clustering of probesets (rows) and samples (columns) with Euclidean distance and complete clustering method. (A) Expression levels in burn patients. (B) Expression levels in trauma patients. (C) Expression levels in septic shock 1 patients. (D) Expression levels in septic shock 2 patients. FIGURE 1 \| Heatmap representation of HERVs in three models of injury. Heatmap of the 25% most variant probesets targeting HERVs in the four datasets: burn, trauma, and 2 septic shock cohorts. Probesets are in rows and samples in columns. Samples are annotated (colored bars on the top) by type of samples (HV in pink, patients in cyan) and day after inclusion (blue scaled). Expression levels are color-coded from blue (low expression) to red (high expression). Similar patterns of expression are highlighted through hierarchical clustering of probesets (rows) and samples (columns) with Euclidean distance and complete clustering method. Frontiers in Immunology \| www.frontiersin.org RESULTS All but one are located in 3 ′ of the LTR33 HERV element targeted by the 230354_at probeset, which is located in the ﬁrst intron of SLC8A1-204 transcript (Figure 3A). The up-modulation of the LTR33 element in septic shock patients observed on microarray was conﬁrmed by RT- qPCR (Figures 2B, 3B). The up-modulation observed for other cohorts was not conﬁrmed by RT-qPCR. The gene SLC8A1 was not expressed in patients or HV, as seen on various microarray probesets and conﬁrmed by RT-qPCR (SLC8A1_gene, var210, var211_212). CD55-211 transcript (Figure 5A). The HERV element overlaps the 3 ′UTR of transcript CD55-211 and a long intergenic noncoding RNA (lincRNA, a class of long transcribed RNA molecules longer than 200 nucleotides and not coding for proteins) (Figure 5B). The up-modulation of MLT1H seen with microarray in the 4 cohorts was partially conﬁrmed by RT- qPCR on trauma and septic shock cohorts (Figures 2B, 5C). The designs targeting MLT1H or close neighborhood (PCR3, 4 and 5) presented the same proﬁle, with a signiﬁcant diﬀerence in septic shock and trauma cohorts compared to HV (PCR4). The design targeting the gene showed also up-modulation of CD55 and a very high absolute normalized expression in patients compared to HV (Figure 5C). [Of note 1555950_a_at probeset, targeting most of CD55 transcripts, was also up-modulated in patients, and with a high expression level (data not shown)]. We also conﬁrmed by ﬂow cytometry on monocytes and neutrophils that CD55 expression was higher in patients than in HV, conﬁrming an up-modulation at the protein level in patients (Figure 5D). SLC8A1 has 11 known transcripts. All but one are located in 3 ′ of the LTR33 HERV element targeted by the 230354_at probeset, which is located in the ﬁrst intron of SLC8A1-204 transcript (Figure 3A). The up-modulation of the LTR33 element in septic shock patients observed on microarray was conﬁrmed by RT- qPCR (Figures 2B, 3B). The up-modulation observed for other cohorts was not conﬁrmed by RT-qPCR. The gene SLC8A1 was not expressed in patients or HV, as seen on various microarray probesets and conﬁrmed by RT-qPCR (SLC8A1_gene, var210, var211_212). NFE4 gene has 2 transcripts (Figure 4A) and only one is coding for a protein (NFE4-202). The LTR101_Mam HERV element, targeted by the 2560527_at probeset, is located in 3 ′UTR of NFE4-201, the non-protein-coding transcript. RESULTS A HERV from MLT1H family (1556107_at) and one from LTR16B2 family (1559777_at) are located in the 3’UTR of CD55 and MIR3945HG genes respectively (Table 2). To validate these transcriptional HERV modulations, we designed primers on the 6 described HERV loci above, and on nearby genes by RT-qPCR (Table S2). For each targeted region, we made multiple RT-qPCR designs. We identiﬁed several distinct patterns of expression comparing HERVs and nearby genes (Tables 2): (i) for PTTG1IP and MIR3945HG regions, we observed no or low signal from the HERV loci (data not shown), (ii) for SLC8A1 (Figure 3) and NFE4 (Figure 4) regions, we observed a high signal from HERVs elements, but no or lower signal on the genes, (iii) for CD55 (Figure 5) and CD300LF (Figure 6) regions, we observed a middle or high signal from both HERV loci and genes. Moreover, we selected 2 other probesets of interest(1553043_a_at and 1560527_at, Figure 2C). The ﬁrst one targets a MLT1D HERV located in the 3 ′UTR of CD300LF. It was up-modulated in burn and SS2 cohorts. It had a strong up-modulation at D1 in burn patients compared To better interpret the results, we extracted from Ensembl the genome annotation and showed in genomic context, the microarray and the RT-qPCR results of SLC8A1 (Figure 3), NFE4 (Figure 4), CD55 (Figure 5) and CD300LF (Figure 6) regions. January 2019 \| Volume 9 \| Article 3091 Frontiers in Immunology \| www.frontiersin.org 5 HERV Modulation in Acute Inﬂammatory Diseases Tabone et al. FIGURE 2 \| Differentially expressed HERVs in severely injured patients. (A) Venn diagram of differentially expressed HERVs for each dataset. (B) Expression proﬁles of commonly modulated probesets targeting HERVs in the 4 datasets, at D1. (C) Expression proﬁles of 2 selected probesets targeting HERVs. Boxes are color-coded by cohort. For each graphic, from top to bottom, title contains: probeset name, HERV name and closest gene. FIGURE 2 \| Differentially expressed HERVs in severely injured patients. (A) Venn diagram of differentially expressed HERVs for each dataset. (B) Expression proﬁles of commonly modulated probesets targeting HERVs in the 4 datasets, at D1. (C) Expression proﬁles of 2 selected probesets targeting HERVs. Boxes are color-coded by cohort. For each graphic, from top to bottom, title contains: probeset name, HERV name and closest gene. SLC8A1 has 11 known transcripts. Frontiers in Immunology \| www.frontiersin.org RESULTS We conﬁrmed the expression of HERV locus, but the up-modulation seen in burn and septic shock 2 cohorts compared to HV on microarray was not conﬁrmed by RT-qPCR, neither for gene nor for HERV designs (Figures 2C, 6C). PCR1 showed no signal at all. PCR2 design showed a slight higher expression level in burn and septic shock cohorts compared to HV. We also conﬁrmed an higher expression at the protein level by ﬂow cytometry on neutrophils in burn and septic shock patients, compared to HV (Figure 6D). In monocytes, protein level in burn patients at D1 seemed slightly higher than HV. or both HERV and 3’UTR of CD300LF (PCR2, Figure 6B). We conﬁrmed the expression of HERV locus, but the up-modulation seen in burn and septic shock 2 cohorts compared to HV on microarray was not conﬁrmed by RT-qPCR, neither for gene nor for HERV designs (Figures 2C, 6C). PCR1 showed no signal at all. PCR2 design showed a slight higher expression level in burn and septic shock cohorts compared to HV. We also conﬁrmed an higher expression at the protein level by ﬂow cytometry on neutrophils in burn and septic shock patients, compared to HV (Figure 6D). In monocytes, protein level in burn patients at D1 seemed slightly higher than HV. Several groups showed that huge epigenetic modiﬁcations occur after acute inﬂammation, regulating transcriptional proﬁles in the immune system, especially in sepsis (34, 35). These epigenetic modiﬁcations may explain the polarization proﬁles such as tolerance or trained immunity, observed after various stimulations of innate cells (35). We hypothesized and conﬁrmed in vivo that other elements than genes, especially HERVs which are known to be tightly controlled by epigenetic modiﬁcations (36), might be modulated in acute inﬂammatory situations. This has also been demonstrated in other pathophysiological contexts such as cancer (7, 8, 37, 38), where global epigenetic modiﬁcations are also observed (39, 40). Frontiers in Immunology \| www.frontiersin.org RESULTS Although the same trends are observed between microarray and RT-qPCR, the up-modulation of the LTR101_Mam element observed in septic shock patients with microarray was not statistically signiﬁcant in RT-qPCR (Figures 2C, 4B). There was low or no signal on designs targeting gene transcripts (NFE4_gene and NFE4_gene_var201). The MLT1D HERV element, targeted by the 1553043_a_at probeset is located in 3 ′UTR of CD300LF-201, 202, 203, 204, and 207 protein-coding transcripts (Figure 6A). We made several RT-qPCR designs, targeting either the HERV locus only (PCR1) CD55 gene has 11 transcripts. The MLT1H HERV element, targeted by the 1556107_at probeset, is located in the 3 ′UTR of January 2019 \| Volume 9 \| Article 3091 Frontiers in Immunology \| www.frontiersin.org 6 HERV Modulation in Acute Inﬂammatory Diseases Tabone et al. TABLE 2 \| Genomic and transcriptomic features of the 6 probesets of interest. HERV Probesetsa Transcriptomic & genomic features 1556107_at 230354_at 1553043_a_at 1560527_at 1559777_at 236982_at Patients vs. HVs UP UP UP (for Burn & SS2) UP (for SS1 & SS2) UP UP log2FCb in Burn 1.13 1.73 1.48 −0.55 1.05 0.77 Trauma 1.31 1.47 −0.33 1.50 0.79 1.57 SS1 0.57 2.03 0.26 0.72 1.07 0.90 SS2 1.45 2.97 1.08 2.00 1.26 1.12 Conﬁrmedc Yes Yes* Yes* Yes* No No HERV family MLT1H LTR33 MLT1D LTR101_Mam LTR16B2 ERV24B_Prim-int HERV coordinatesd chr1 207372720- 207272854 chr2 40545338-40545778 chr17 74694268-74694744 chr7 102988743- 102988923 chr4 184844993- 184845324 chr21 44875454-44876122 Closest gene CD55 SLC8A1 CD300LF NFE4 MIR3945HG PTTG1IP Localizatione 3 ′ UTR intron 1 3 ′UTR 3 ′end 3 ′ UTR promoter region aHGU133plus2 Affymetrix probesets mapping on a HERV locus. bA positive log2 FC means that the probeset is more expressed in patients than in HV. cConﬁrmed by RT-qPCR. Expression conﬁrmed. Modulation between patients and HV not always statistically conﬁrmed, mainly due to high inter-individual variability. dGrch38 genomic coordinates of HERV locus. eLocalization of HERV element according to the nearby gene. TABLE 2 \| Genomic and transcriptomic features of the 6 probesets of interest. Genomic and transcriptomic features of the 6 probesets of interes Moreover, very few data are available in human diseases for speciﬁc loci. No study had yet evaluated the expression of HERVs in acute inﬂammatory contexts by using multiple cohorts with diﬀerent types of inﬂammatory injuries. or both HERV and 3’UTR of CD300LF (PCR2, Figure 6B). DISCUSSION Statistically signiﬁcant difference with HV is marked by (Wilcoxon signed rank test, p < 0.05). FIGURE 4 \| LTR101_Mam HERV and NFE4 gene expression. (A) NFE4 genomic region, with the position of HERV in green, of probeset in dark blue, of PCR designs in purple. (B) Expression levels of speciﬁc transcripts by RT-qPCR, as described in A, in HV and patients at D1. Expression levels (copy number / µl) were normalized with reference gene (HPRT1). Boxes are color-coded by cohort. Statistically signiﬁcant difference with HV is marked by * (Wilcoxon signed rank test, p < 0.05). FIGURE 3 \| LTR33 HERV and SLC8A1 gene expression. (A) SLC8A1 genomic region, with the position of HERV in green, probeset in dark blue, and PCR designs in purple. (B) Expression levels of speciﬁc transcripts by RT-qPCR, as described in A, in HV and patients at D1. Expression levels (copy number / µl) were normalized with reference gene (HPRT1). Boxes are color-coded by cohort. Statistically signiﬁcant difference with HV is marked by * (Wilcoxon signed rank test, p < 0.05). FIGURE 4 \| LTR101_Mam HERV and NFE4 gene expression. (A) NFE4 genomic region, with the position of HERV in green, of probeset in dark blue, of PCR designs in purple. (B) Expression levels of speciﬁc transcripts by RT-qPCR, as described in A, in HV and patients at D1. Expression levels (copy number / µl) were normalized with reference gene (HPRT1). Boxes are color-coded by cohort. Statistically signiﬁcant difference with HV is marked by * (Wilcoxon signed rank test, p < 0.05). FIGURE 3 \| LTR33 HERV and SLC8A1 gene expression. (A) SLC8A1 genomic region, with the position of HERV in green, probeset in dark blue, and PCR designs in purple. (B) Expression levels of speciﬁc transcripts by RT-qPCR, as described in A, in HV and patients at D1. Expression levels (copy number / µl) were normalized with reference gene (HPRT1). Boxes are color-coded by cohort. Statistically signiﬁcant difference with HV is marked by * (Wilcoxon signed rank test, p < 0.05). targeting HERVs were initially supposed to target conventional genes, the majority of explored HERVs are close to or within a gene. To better understand HERV expression in these settings, targeting HERVs localized far from genes seems important. Until now, the lack of tool made diﬃcult the exploration of HERV expression. Frontiers in Immunology \| www.frontiersin.org January 2019 \| Volume 9 \| Article 3091 DISCUSSION We took advantage of previous microarray analyses on four cohorts of severely injured patients to assess the modulation of HERV transcriptome in acute inﬂammation. We showed that several loci were expressed and modulated after acute injury. Surprisingly, a large majority among the modulated HERVs were down-modulated in patients compared to HV, whereas a global and massive gene up-modulation has been observed after severe injuries (17). Interestingly in cancer, epigenetic modiﬁcations that gave access to HERV cis sequences through open chromatin, have also revealed a very role in pathophysiology (2, 5, 37). Indeed, by providing alternative promoter sequences to classical protein coding genes, these epigenetic modiﬁcations explain part of the ectopic expression of myeloid-growth factor receptors in lymphoid cells (37). Recently, it has been suggested that HERV could provide transcription factor binding sites, modulating immune-related gene expression, as illustrated by MER41 elements which bring STAT1 motifs to IFNγ inducible genes (41). An exhaustive study on HERV expression with a diﬀerent tool, like CHIPseq technology, would bring valuable data to ﬁnd potential TFBS on HERV sequences. This underlines how HERV elements, in particular their LTRs, could modulate gene expression and the host immune response to injury. In our study, the four commonly modulated HERVs were LTRs located nearby genes related to the immune response. In several cases (NFE4, Five HERVs were modulated in patients compared to HV in all four datasets and 16 HERVs in at least 3 datasets, suggesting a similar inﬂammatory triggered modulation in all models. We validated expression proﬁles by RT-qPCR on 6 regions, allowing us to explore more precisely the modulation pattern of the HERVs and the neighbor genes. Interestingly, all these 6 HERVs have detected signals in RNAseq experiments from lymphoid cells and whole blood datasets [Ensembl Rnaseq tracks (33)]. Some authors already focused on HERV detection in blood of burn patients using pan-family RT-PCRs (14, 15). January 2019 \| Volume 9 \| Article 3091 7 HERV Modulation in Acute Inﬂammatory Diseases Tabone et al. FIGURE 3 \| LTR33 HERV and SLC8A1 gene expression. (A) SLC8A1 genomic region, with the position of HERV in green, probeset in dark blue, and PCR designs in purple. (B) Expression levels of speciﬁc transcripts by RT-qPCR, as described in A, in HV and patients at D1. Expression levels (copy number / µl) were normalized with reference gene (HPRT1). Boxes are color-coded by cohort. DISCUSSION (C) Expression levels of speciﬁc transcripts by RT-qPCR, as described in A and B, in HV and patients at sion levels (copy number/µl) were normalized with reference gene (HPRT1). Boxes are color-coded by cohort. (D) Protein expression levels (MFI), on cytes (left) and neutrophils (right) from 8 burn patients (red), 11 septic shock patients (blue), and 9 HV (purple). Columns ISO B, ISO SS, and ISO HV corresp ypes for burn, septic shock, and HV, respectively. Statistically signiﬁcant difference with HV is marked by * (Wilcoxon signed rank test, p < 0.05). h blood-cell type to HERV expression and in another how HERV expression may contribute to the cell time that speciﬁc HERV loci are transcribed in whole b of ICU patients. Our design allowed us to identify spe RE 5 \| CD55 associated HERV. (A) CD55 genomic region, with the positions of HERV in green, of probeset in dark blue, of PCR designs in purple. (B) Zoom in mic region of HERV showing PCR designs in detail. (C) Expression levels of speciﬁc transcripts by RT-qPCR, as described in A and B, in HV and patients at D1. ession levels (copy number/µl) were normalized with reference gene (HPRT1). Boxes are color-coded by cohort. (D) Protein expression levels (MFI), on ocytes (left) and neutrophils (right) from 8 burn patients (red), 11 septic shock patients (blue), and 9 HV (purple). Columns ISO B, ISO SS, and ISO HV correspond types for burn, septic shock, and HV, respectively. Statistically signiﬁcant difference with HV is marked by * (Wilcoxon signed rank test, p < 0.05). ch blood-cell type to HERV expression and in another how HERV expression may contribute to the cell i li T l d h d f h ﬁ time that speciﬁc HERV loci are transcribed in whole blood of ICU patients. Our design allowed us to identify speciﬁc i i l i f HERV l i i li k d FIGURE 5 \| CD55 associated HERV. (A) CD55 genomic region, with the positions of HERV in green, of probeset in dark blue, of PCR designs in purple. (B) Zoom in genomic region of HERV showing PCR designs in detail. (C) Expression levels of speciﬁc transcripts by RT-qPCR, as described in A and B, in HV and patients at D1. Expression levels (copy number/µl) were normalized with reference gene (HPRT1). Boxes are color-coded by cohort. DISCUSSION It would be interesting to reproduce these analyses, with a more exhaustive technology designed to speciﬁcally target HERVs, like the HERV-V3 Aﬀymetrix microarray we recently published (43), or even RNAseq. It will allow us to better describe the whole HERV transcriptome modulation and understand the putative global role of HERV in the host response. CD300LF), we found a polyadenylation signal (AAUAAA) provided by the HERV LTR in 3 ′ of some of the alternative transcripts of the genes. The case of CD300LF is interesting as this protein acts as an inhibitory receptor for myeloid cells (42). The LTR might stabilize speciﬁc transcripts and enhance expression of CD300LF protein, which we conﬁrmed by ﬂow cytometry in severe burn patients early after admission. This up regulation might participate in the compensatory anti-inﬂammatory response. The precise understanding of the mechanisms through which speciﬁc HERV LTRs might impact immune gene expression is not possible in such translational research setting with patient samples. This will require in the future in vitro experimental models to validate and understand our observations. Finally, it would be of importance to consider HERV expression in further blood transcriptome analyses, especially in such acute inﬂammatory contexts, to better understand HERV expression during host response. Such studies, based on well- deﬁned cohorts including criteria for patient stratiﬁcation and taking into account drug treatment, should allow to estimate whether HERVs could be good markers of the diﬀerent immune phases that follow acute injury. More, whether HERV could become potential therapeutic targets would basically require to decipher which circulating cell type produces which HERV. It will thus be appropriate to develop dedicated cellular models to, in one hand better understand the contribution Our RT-qPCR validation assays also showed inter-individual variability and underlined that exploring such repertoire of our genome, repetitive sequences, may face speciﬁcity issues, and will require speciﬁc tools. Indeed, as a ﬁrst attempt, we used commercial microarray where probesets were not initially designed to target HERV elements. Moreover, as the probesets January 2019 \| Volume 9 \| Article 3091 8 HERV Modulation in Acute Inﬂammatory Diseases Tabone et al. et al. HERV Modulation in Acute Inﬂammatory Dis RE 5 \| CD55 associated HERV. (A) CD55 genomic region, with the positions of HERV in green, of probeset in dark blue, of PCR designs in purple. (B) Zoom mic region of HERV showing PCR designs in detail. DISCUSSION Columns ISO B, ISO SS, and ISO HV ond to isotypes for burn septic shock and HV respectively Statistically signiﬁcant difference with HV is marked by * (Wilcoxon signed rank test p < 0 05) FIGURE 6 \| CD300LF associated HERV. (A) CD300LF genomic region, with the positions of HERV in green, of probeset in dark blue, of PCR designs in purple. (B) Zoom in genomic region of HERV showing PCR designs in detail. (C) Expression levels of speciﬁc transcripts by RT-qPCR, as described in A and B, in HV and patients at D1. Expression levels (copy number/µl) were normalized with reference gene (HPRT1). Boxes are color-coded by cohort. (D) Protein expression levels (MFI), on monocytes (left), and neutrophils (right) from 14 burn patients (red), 11 septic shock patients (blue), and 10 HV (purple). Columns ISO B, ISO SS, and ISO HV correspond to isotypes for burn, septic shock, and HV, respectively. Statistically signiﬁcant difference with HV is marked by * (Wilcoxon signed rank test, p < 0.05). DISCUSSION (D) Protein expression levels (MFI), on monocytes (left) and neutrophils (right) from 8 burn patients (red), 11 septic shock patients (blue), and 9 HV (purple). Columns ISO B, ISO SS, and ISO HV correspond to isotypes for burn, septic shock, and HV, respectively. Statistically signiﬁcant difference with HV is marked by * (Wilcoxon signed rank test, p < 0.05). time that speciﬁc HERV loci are transcribed in whole blood of ICU patients. Our design allowed us to identify speciﬁc transcriptional signatures of HERVs elements, in vivo, linked of each blood-cell type to HERV expression and in another hand how HERV expression may contribute to the cell response to stimuli. To conclude, we showed for the ﬁrst January 2019 \| Volume 9 \| Article 3091 Frontiers in Immunology \| www.frontiersin.org 9 HERV Modulation in Acute Inﬂammatory Diseases Tabone et al. GURE 6 \| CD300LF associated HERV. (A) CD300LF genomic region, with the positions of HERV in green, of probeset in dark blue, of PCR designs in purple. (B) oom in genomic region of HERV showing PCR designs in detail. (C) Expression levels of speciﬁc transcripts by RT-qPCR, as described in A and B, in HV and atients at D1. Expression levels (copy number/µl) were normalized with reference gene (HPRT1). Boxes are color-coded by cohort. (D) Protein expression levels MFI), on monocytes (left), and neutrophils (right) from 14 burn patients (red), 11 septic shock patients (blue), and 10 HV (purple). Columns ISO B, ISO SS, and ISO HV orrespond to isotypes for burn, septic shock, and HV, respectively. Statistically signiﬁcant difference with HV is marked by * (Wilcoxon signed rank test, p < 0.05). E 6 \| CD300LF associated HERV. (A) CD300LF genomic region, with the positions of HERV in green, of probeset in dark blue, of PCR designs in purple. (B) genomic region of HERV showing PCR designs in detail. (C) Expression levels of speciﬁc transcripts by RT-qPCR, as described in A and B, in HV and at D1. Expression levels (copy number/µl) were normalized with reference gene (HPRT1). Boxes are color-coded by cohort. (D) Protein expression levels n monocytes (left), and neutrophils (right) from 14 burn patients (red), 11 septic shock patients (blue), and 10 HV (purple). ACKNOWLEDGMENTS Authors would like to gratefully thank Maria-Paola Pisano, Marie-Angélique Cazalis, Boris Meunier, Julie Mouillaux and Estelle Peronnet for their kind advices. They also thank all clinical research assistant for the collection of blood samples, especially Hélène Vallin and Valérie Cerro. Finally they gratefully thank Anne Portier and Marie-Angélique Cazalis for all experiments made on samples. Supplemental Figure 5 \| Differentially expressed HERVs in severely injured patients. (A) Venn diagram of differentially expressed HERVs for each dataset. (B) Expression proﬁles of commonly modulated probesets targeting HERVs in the 4 datasets. Boxes are color-coded by day after inclusion. (C) Expression proﬁles of 2 selected probesets targeting HERVs. For each graphic, from top to bottom, title contains: probeset name, HERV name and closest gene. Table S1 \| Clinical description of trauma cohort. Table S1 \| Clinical description of trauma cohort. FUNDING Supplemental Figure 3 \| Volcano plots of differentially expressed HERVs. (A) in burn cohort. (B) in trauma cohort. (C) in septic shock cohort 1 and (D) in septic shock cohort 2. The x-axis represents the log2 fold change between patient and HV, the y-axis the –log10 of adjusted p-values. Each point represents a probeset targeting HERV, in red the statistically differentially expressed between patients at D1 and HV. On each volcano plot, the number indicates the number of differentially expressed probesets. This work was supported by bioMerieux SA and Hospices Civils de Lyon. MM and OT were supported by doctoral grants from bioMerieux. In addition, OT was supported by the Association Nationale de la Recherche et de la Technologie (ANRT), convention N◦2015/1227. This study received funding from bioMérieux and Hospices Civils de Lyon, and was part of ADNA (Advanced Diagnostic for New Therapeutic Approaches), a program dedicated to personalized medicine, coordinated by Institut Mérieux and supported by French public agency BPI France. Supplemental Figure 4 \| Heatmap representation of the modulated HERVs in severely injured patients at D1. Heatmap of the 56 differentially expressed probesets in at least 1 dataset. On the top bar, samples are color-coded in blue for HV and in red for Patients. On the bar below, samples are in green for Burn study, in yellow for Trauma study, in purple for Septic Shock 1 (SS1) study and in light red for Septic Shock 2 (SS2). Probesets are in rows and samples in columns. Expression levels from each cohort have been normalized (centered and reduced). Normalized expression levels are color-coded from blue (low expression) to red (high expression). Similar patterns of expression are highlighted through hierarchical clustering of probesets (rows) and samples (columns) with Euclidean distance and complete clustering method. DATA AVAILABILITY STATEMENT to the acute inﬂammatory response. Moreover, the similarities observed in three models of acute injuries suggest common regulatory mechanisms and a speciﬁcity of the observed modulation. We also unravel the potential regulatory role of these elements within the host immune response. Further studies are needed to better understand such mechanisms and how HERVs may contribute to the pathophysiology of the host immune response, a key part of the pathophysiology of sepsis. Microarray expression data has been deposited on NCBI Gene Expression Omnibus and are accessible through GEO accession numbers GEO:GSE77791, GEO:GSE57065 and GEO:GSE95233. Data from microarray experiment for trauma cohort are available at Hospices Civils de Lyon—bioMérieux—UCBL1 “Pathophysiology of Injury Induced Immunosuppression,” Groupement Hospitalier Edouard Herriot, France. Microarray expression data has been deposited on NCBI Gene Expression Omnibus and are accessible through GEO accession numbers GEO:GSE77791, GEO:GSE57065 and GEO:GSE95233. Data from microarray experiment for trauma cohort are available at Hospices Civils de Lyon—bioMérieux—UCBL1 “Pathophysiology of Injury Induced Immunosuppression,” Groupement Hospitalier Edouard Herriot, France. January 2019 \| Volume 9 \| Article 3091 Frontiers in Immunology \| www.frontiersin.org Frontiers in Immunology \| www.frontiersin.org 10 HERV Modulation in Acute Inﬂammatory Diseases Tabone et al. SUPPLEMENTARY MATERIAL Table S2 \| Designs of qPCR systems. (A) Home-made designs. (B) Commercial designs. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ﬁmmu. 2018.03091/full#supplementary-material Table S3 \| Differentially expressed HERVs. The 56 differentially expressed probesets targeting HERVs between patients at D1 and HV, in at least 1 dataset. Each tab correspond to a dataset. Each table shows the probeset names, the log2 fold changes, the Average Expression, the moderated t-statistic, the raw p-value, the adjusted p-value and the log-odds that the gene is differentially expressed. Table S3 \| Differentially expressed HERVs. The 56 differentially expressed probesets targeting HERVs between patients at D1 and HV, in at least 1 dataset. Each tab correspond to a dataset. Each table shows the probeset names, the log2 fold changes, the Average Expression, the moderated t-statistic, the raw p-value, the adjusted p-value and the log-odds that the gene is differentially expressed. Supplemental Figure 1 \| Heatmap representation of HERVs in three models of injury. Heatmap of the 337 probesets targeting HERVs in the four datasets: burn, AUTHOR CONTRIBUTIONS trauma and 2 septic shock cohorts. Probesets are in rows and samples in columns. Samples are annotated (colored bars on the top) by type of samples (HV in pink, patients in cyan) and day after inclusion (blue scaled). Expression levels are color-coded from blue (low expression) to red (high expression). Similar patterns of expression are highlighted through hierarchical clustering of probesets (rows) with Euclidean distance and complete clustering method. (A) Expression levels in burns. (B) Expression levels in traumas. (C) Expression levels in septic shock 1. (D) Expression levels in septic shock 2. On each heatmap, the percentage of probesets with low intensity is shown. trauma and 2 septic shock cohorts. Probesets are in rows and samples in columns. Samples are annotated (colored bars on the top) by type of samples (HV in pink, patients in cyan) and day after inclusion (blue scaled). Expression levels are color-coded from blue (low expression) to red (high expression). Similar patterns of expression are highlighted through hierarchical clustering of probesets (rows) with Euclidean distance and complete clustering method. (A) Expression levels in burns. (B) Expression levels in traumas. (C) Expression levels in septic shock 1. (D) Expression levels in septic shock 2. On each heatmap, the percentage of probesets with low intensity is shown. OT, FM and JT designed the project, performed the analyses and wrote the paper. CJ and FV performed cytometry experiments. EC performed RT-qPCR validations. ML, AL, BA, and TR recruited patients in the various cohorts. OT, MM, FV, FM, and JT read and discussed the manuscript. All authors drafted or revised critically the manuscript for important intellectual contents. 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W4311688511.txt	https://bmccardiovascdisord.biomedcentral.com/counter/pdf/10.1186/s12872-022-03002-z	en		Sex differences involved in persistent atrial fibrillation recurrence after radiofrequency ablation	BMC cardiovascular disorders	2,022	cc-by	4,574	(2022) 22:549 Li et al. BMC Cardiovascular Disorders https://doi.org/10.1186/s12872-022-03002-z Open Access RESEARCH Sex differences involved in persistent atrial fibrillation recurrence after radiofrequency ablation Haiwei Li1, Zefeng Wang2, Zichao Cheng1, Yingming Zhu3, Zhongyu Yuan1, Jianwei Gao1,4, Xiaoping Zhang1,4,5* and Yongquan Wu2* Abstract Background: In recent years, the difference in outcomes of radiofrequency catheter ablation (RFCA) in persistent atrial fibrillation patients has risen. In particular, biological sex seems involved in a different response to the AF ablation procedure. In our study, we analyzed the AF recurrences after RFCA assessing the other association between male/female patients with the outcomes. Methods: We enrolled 106 patients (74.5% men) with persistent atrial fibrillation with scheduled follow-up. The baseline clinical characteristics and AF recurrence after RFCA were compared between men and women. Cox regression analyses were performed to determine the risk predictors of AF recurrence. Results: The proportion of RFCA in women was lower than that in men. Men with persistent AF were younger than women (58.6 ± 10.4 years vs. 65.1 ± 8.7 years, respectively; p = 0.003). The left atrium (LA) diameter was higher in males (43.7 ± 4.6 mm vs. 41.3 ± 5.5 mm; p = 0.028), and the level of left heart ejection fraction (LVEF) was higher in females (59.4 ± 6.9% vs. 64.1 ± 5.5%; p = 0.001). Sex differences in AF recurrence after RFCA were significant during the median 24.4-month (interquartile range: 15.2–30.6 months) follow-up period, and the recurrence rate of AF in women was significantly higher than that in men (p = 0.005). Univariable Cox regression analysis showed that female sex was a risk factor for persistent AF recurrence after RFCA [HR: 2.099 (1.087–4.053)]. Univariate Cox regression analysis revealed that non-PV ablation not associated with AF recurrence [HR: 1.003 (0.516–1.947)]. Conclusion: In a monocentric cohort of persistent AF patients, the female biological sex was associated with a higher risk of AF recurrence after RFCA. Keywords: Sex differences, Persistent atrial fibrillation, Radiofrequency ablation, Recurrence Correspondence: xiaoping_zhang2004@ccmu.edu.cn; wuyongquan67@163. com 1 Beijing Anzhen Hospital, Capital Medical University, Beijing, People’s Republic of China 2 Department of Cardiology, Beijing Anzhen Hospital, Capital Medical University, No. 2 Anzhen Road, Chaoyang District, Beijing 100029, People’s Republic of China Full list of author information is available at the end of the article Background Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia worldwide. Recent studies have shown that the estimated prevalence of AF in adults is between 2 and 4%. AF occurrence associated with variety risk factors including demographic factors, health factors and other clinical diseases [1–3]. Similar to other cardiovascular diseases, sex differences exist in patients with AF. Previous studies have demonstrated that the incidence of AF is higher in men than in women. The onset age of AF © The Author(s) 2022. 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://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Li et al. BMC Cardiovascular Disorders (2022) 22:549 in men is higher than that in women, but women with AF have a greater symptom burden [4]. Additionally, women undergo ablation less frequently than men, although they typically have a greater symptom burden [5]. Studies of AF catheter ablation have also shown significant sexbased differences, including a higher risk of AF recurrence rate and increased periprocedural complications and hospitalization in women compared with men [5, 6]. Despite being extensively researched, sex-based differences remain poorly understood. Until now, whether these differences are due to sex-based pathophysiological differences, differences in clinical characteristics, or unreasonable bias has been unclear. Additionally, AF management and long-term outcomes seem to be significant across countries. In the current study, we evaluated sex-based differences in persistent AF, focusing on comorbidities, clinical presentation, and laboratory examination. We also sought to identify predictors of the long-term recurrence of AF. Methods Design and sample selection Between January 2019 and December 2020, consecutive patients aged ≥ 18 years who underwent initial RFCA in the Department of Cardiology of Beijing AnZhen Hospital were reviewed. The inclusion criteria were as follows: (1) patients > 18 years of age; (2) patients were diagnosed persistent AF and underwent first RFCA treatment; (3) signed informed consent at admission. The exclusion criteria were: (1) presence of other cardiovascular disease, including abnormal cardiac structures disease, heart failure, and history of CABG (coronary artery bypass grafting), RFCA(radiofrequency catheter ablation), and other cardiac surgical; (2) Incomplete medical history or follow-up data; (3) Other disease including mental disease, advanced malignant tumor, acute, severe renal dysfunction and chronic inflammatory diseases, and autoimmune diseases; (4) Pregnancy; (5) LA anteroposterior diameter > 50 mm. The study was designed and performed in accordance with the Declaration of Helsinki for Human Research and was approved by the Beijing Anzhen Hospital Ethics Committee (Approval NO: 2022042X). Data collection Clinical and laboratory data of all patients were collected, which are including: (1) general clinical data: age, gender, body mass index (BMI, kg/m2), comorbidities and calculation of EHRA score, CHA2DS2-VASc score, HAS-BLED score, history of medication and echocardiographic parameters (Echocardiography were performed with a Philips 7C color Doppler ultrasound); (2) Laboratory examinations (results of fasting blood sample Page 2 of 7 obtained on the latest preoperative morning): white blood cell, red blood cell, platelet count, hemoglobin, estimated Glomerular filtration rate, fasting plasma glucose, homocysteine, uric acid, B-type natriuretic peptide, alanine aminotransferase, aspartate transaminase, lactate dehydrogenase, albumin, total bilirubin, total cholesterol, low-density lipoprotein cholesterol. Radiofrequency catheter ablation (RFCA) procedure All patient underwent anticoagulation administration and transesophageal echocardiography (TEE) to exclude atrial thrombus before RFCA. Catheter ablation procedures were performed after obtain patient agreement form to treatment signing. CPVI (circumferential pulmonary vein isolation) were performed for all participants and selective additional atrial ablation (ie, non-pulmonary veins foci ablation) was only performed in patients with AF persisting despite completion of CPVI. Point-bypoint CPVI was performed using irrigated ablation catheters in a power control mode at 35 W (irrigation flow 17 mL/min). The ablation index (AI) had a target value of 500 for the anterior LA wall and 400 for the posterior wall. Intravenous heparin was administered continuously to maintain an activated clotting time between 250 and 350 s after transseptal puncture. an irrigation catheter was used in combination with a 3-dimensional mapping system (CARTO, Biosense Webster) conducted using PentaRay. The electrophysiological end point of CPVI was a bidirectional conduction block between the left atrium (LA) and PVs. Follow‑up Antiarrhythmic and anticoagulant treatments were maintained for at least 3 months after RFCA. Periodic followup visits were conducted at 3, 6, 12, 24 and 36 months in outpatient clinics. Each visit includes medical and physical examination, 12-lead electrocardiogram (ECG) and 24 h Holter monitoring. The endpoint was AF recurrence defined as any documented atrial tachyarrhythmia (atrial fibrillation, atrial flutter, or atrial tachycardia) episode lasting for at least 30 s after RFCA, excluding 3-month blanking period. Follow-up records were based on telephone interviews, and patients’ preiodic visits to outpatient clinics at the Beijing Anzhen Hospital and outcomes were judged by trained study personnel and cardiologists. They were strongly recommended to visit the closest hospital for an ECG if they felt discomfort due to cardiac arrhythmia. Statistical analysis Participants were divided into 2 groups according to gender. Continuous variables with normal distribution are expressed as mean ± standard deviation (SD), and Li et al. BMC Cardiovascular Disorders (2022) 22:549 Page 3 of 7 non-normally distributed variables were described as median and interquartile range (IQR). Comparisons of means between groups were analyzed using the independent sample t-test for normally distributed data and the Mann–Whitney test for non-normally distributed data. Categorical data were presented as frequencies or percentages and compared between groups using the chi-squared test or Fisher’s exact test. Univariate Cox regression analyses were performed to determine risk factors for persistent AF recurrence, and the hazard ratio (HR) and 95% CI were calculated. Adjusted model were done based age, comorbidities, BMI, LVEF, EHRA score, CHA2DS2-VASc score, and HAS-BLED score to exclude confounding factors. Time-dependent survival between groups (male and female) was evaluated using Kaplan– Meier curves and the Log-rank test. All data were analyzed with SPSS 20.0 (IBM Corp., Armonk, NY, USA), R (version 4.0.4). Two-tailed values of p < 0.05 were considered statistically significant. Results Baseline clinical characteristics A total of 136 patients were included in this study; 24 patients were lost to follow-up, and 6 patients with incomplete clinical information were excluded (Fig. 1). As shown in Table 1, 106 consecutive persistent AF patients (aged 60.4 ± 10.3 years) were recruited, 77 men (aged 58.6 ± 10.4 years) and 29 women (aged 65.1 ± 8.7 years). The proportion of men undergoing RFCA was higher than that of women, and the age of female patients with RFCA was significantly higher than that of male patients (p = 0.003). The proportion of women with CHA2DS2VASc scores ≥ 2 was significantly higher than that of men (Table 1) (p < 0.05). The LA diameter was higher in men (43.7 ± 4.6 mm vs. 41.3 ± 5.5 mm; p = 0.028), and the level of LVEF was higher in women (59.4 ± 6.9% vs. 64.1 ± 5.5%; p = 0.001). Significant sex differences in AF recurrence after RFCA procedures were found, and the recurrence rate of persistent AF in women was significantly higher than that in men (p = 0.005). The levels of RBC and Hb in the male group were significantly higher than those in the female group (Additional file 2: Table S2) (p < 0.05). No significant differences were found in other clinical characteristics, including the body mass index (BMI), medical history, the EHRA score, the HASBLED score, and laboratory examination, between the male and female groups (all p > 0.05). Clinical outcomes The median follow-up time after RFCA was 24.4 months (interquartile range: 15.2–30.6 months), and 36 patients showed recurrence (20 men and 16 women) after AF ablation during the follow-up period. Kaplan–Meier Fig. 1 Flow diagram showing population selection and study design survival analysis showed that the 12-month free from persistent AF recurrence rate was 81.7%, 86.7% for men and 72.4% for women (Additional file 1: Figure S1 and Fig. 2). Univariate Cox regression analysis revealed that non-PV ablation not associated with AF recurrence [HR: 1.003(0.516–1.947); p = 0.994]. Univariate Cox regression analysis showed that female sex was a risk factor for persistent AF recurrence after RFCA (Table 2) [HR: 2.099 (1.087–4.053); p = 0.027]. The unadjusted models revealed the relationship between sex differences and persistent AF recurrence, and the proportion of women with increasing persistent AF recurrence was unchanged after model adjustment [HR: 2.371 (1.078–5.215); p = 0.032, as shown in Table 3]. Discussions Our study revealed the following: (1) the proportion of RFCA in women was lower than that in men. (2) The risk of AF recurrence was significantly higher in women than in men. (3) Female biological sex was associated with the risk factor for AF recurrence at univariable Cox regression analysis. Previous trails have revealed that heart failure, renal dysfunction and health status have an impact on AF Li et al. BMC Cardiovascular Disorders (2022) 22:549 Page 4 of 7 Table 1 Baseline clinical characteristics of the persistent AF stratified according to gender Characteristic Total (106) Male (77) Female (29) Age, years 60.4 ± 10.3 58.6 ± 10.4 65.1 ± 8.7 0.003 43.0 ± 46.6 43.6 ± 48.6 41.4 ± 41.5 0.831 BMI, kg/m2 AF duration, months Medical history 27.1 ± 3.9 27.3 ± 4.0 26.6 ± 3.8 p value 0.398 CAD, n (%) 12 (11.3) 10 (13) 2 (6.9) 0.38 HTN, n (%) 64 (60.4) 49 (63.6) 15 (51.7) 0.266 DM, n (%) 15 (14.2) 13 (16.9) 2 (6.9) 0.191 stroke, n (%) 11 (10.4) 6 (7.8) 5 (17.2) 0.157 Medical therapy at admission Statins, n (%) 24 (22.6) 20 (26) 4 (13.8) 0.184 ACEI/ARB, n (%) 23 (21.7) 15 (19.5) 8 (27.6) 0.369 CCB, n (%) 21 (19.8) 14 (18.2) 7 (24.1) 0.495 Beta-block, n (%) 22 (19.8) 17 (22.1) 5 (17.2) 0.586 12 (11.3) 8 (10.4) 4 (13.8) AF related score EHRA score 1, n (%) 0.886 2, n (%) 58 (54.7) 43 (55.8) 15 (51.7) 3, n (%) 36 (34) 26 (33.8) 10 (34.5) 57 (53.8) 50 (64.9) 11 (37.9) < 0.001 CHA2DS2-VASc score 0 or 1, n (%) ≥ 2, n (%) HAS-BLED score ≥ 3, n (%) 49 (46.2) 27 (35.1) 18 (62.1) 4 (3.8) 3 (3.9) 1 (3.4) 0.915 Echocardiographic parameters LAd, mm LVEF, % 43.0 ± 5.0 60.7 ± 6.8 43.7 ± 4.6 59.4 ± 6.9 41.3 ± 5.5 64.1 ± 5.5 0.028 0.001 Non-PV ablation, n (%) 61 (57.5) 47 (61) 14 (48.3) 0.238 Recurrence, n (%) 36 (34) 20 (26) 16 (55.2) 0.005 BMI, Body mass index; CAD, coronary artery disease; HTN, hypertension; DM, diabetes mellitus; ACEI, angiotensin-converting enzyme inhibitors; ARB, angiotensin receptor blocker; CCB, calcium channel blocker; LAd, Left atrium diameter; LVEF, left ventricular ejection fraction recurrence after RFCA [7, 8]. To reduce the impact of comorbidities for our study, we excluded these patients in our exclusion criteria. Initially, the total numbers of persistent AF in our study are 136, and 24 patients were lost to follow-up, 6 patients with incomplete clinical information were excluded. Finally, 106 persistent AF were included in our study to analyze AF recurrence in genders after meeting the inclusion and exclusion criteria. Sex-related differences in the epidemiology of the AF population have been reported in many studies. Women with AF are older, have a higher heart rate, have more symptoms, have a poorer quality of life and have a worse prognosis, with an increasing prevalence of stroke and death [9–11]. Similarly, our study demonstrated that female sex patients were older than male sex patients at the time of RFCA (65.1 ± 8.7 vs. 58.6 ± 10.4, respectively; p = 0.003), and women had higher CHA2DS2-VASc scores, which correspond to a history of stroke/transient ischemic attack. Previous studies have disclosed that men have higher incidence rates of AF than women, and these differences may be related to sex-dependent physiological and pathophysiological mechanisms [12, 13]. Consistent with previous studies, we revealed that the LA diameter was larger in men than in women (43.7 ± 4.6 mm vs. 41.3 ± 5.5 mm, respectively; p = 0.028) and that LVEF was lower in men. A larger LA diameter and lower LVEF in men have been associated with an increased risk of AF and may also explain the lower incidence of AF among women. Current studies on the outcomes of RFCA for atrial fibrillation in women are contradictory. Some studies have revealed that the AF recurrence rate was similar in women and men [14–16]. However, other studies have shown that women had lower arrhythmia-free survival following ablation than men [17, 18]. In our study, the risk of persistent atrial fibrillation recurrence after RFCA in women was significantly higher than that in men. Li et al. BMC Cardiovascular Disorders (2022) 22:549 Page 5 of 7 Fig. 2 Kaplan–Meier survival curves for Freedom from AF/AT recurrence stratified by gender The exact mechanisms for sex-related differences in AF recurrence after the procedure remain incompletely understood. Among patients with AF, women may have more severe and complex arrhythmogenic substrates than men. Pak et al. reported that the LA dimension, LA voltage and pericardial fat volume were lower in women than in their male counterparts [19]. A recent study found that low voltage areas in the atrium can reflect atrial fibrotic remodeling, leading to atrial fibrillation, and the presence of low voltage zones was also a predictor for AF recurrence after catheter ablation [20]. However, Takigawa et al. revealed that the prevalence of non-pulmonary vein foci was significantly more frequent in women than in men (p < 0.05) [21]. Research also disclosed that compared with typical PV foci, nonPV foci are easily associated with AF recurrence after catheter ablation [22]. Therefore, non-PV foci may play a crucial role in the sex difference for AF recurrence after RFCA. However, we found that non-PV ablation not associated with gender and AF recurrence in our study. In the future, ablation of non-PV foci may be critical for maintaining sinus rhythm in women, potentially resulting in additional benefits for women and more research is needed to verify. Study limitations Firstly, our study included persistent AF patients after RFCA and was a single-center study; therefore, the results may be prone to selection bias or information bias. Secondly, the AF patients included in this study were small samples; therefore, the study may have been underpowered to detect some real differences between groups. Thirdly, 24‐h Holter monitoring may lead to an underestimation of the recurrence rates compared with the implanted loop recorder. Fourthly, the results of this study may have been influenced by the inclusion and exclusion criteria, operator experience, and varying techniques used by the operators. Therefore, the generalizability of the results to other populations is unclear. Conclusions In a monocentric cohort of persistent AF patients, the female biological sex was associated with a higher risk of AF recurrence after RFCA. Li et al. BMC Cardiovascular Disorders (2022) 22:549 Page 6 of 7 Table 2 Univariate Cox regression analysis of factors related to persistent AF recurrence Characteristic HR (95%CI) p value Age 1.006 (0.976–1.037) 0.695 Gender (female vs. male) 2.099 (1.087–4.053) 0.027 BMI 1.003 (0.926–1.087) 0.936 AF duration 1.006 (0.999–1.013) 0.089 CAD 0.719 (0.22–2.351) 0.585 HTN 1.2 (0.608–2.37) 0.599 DM 0.689 (0.243–1.952) 0.483 stroke 1.37 (0.53–3.539) 0.516 Statins 0.9 (0.41–1.975) 0.792 ACEI/ARB 1.325 (0.623–2.82) 0.465 CCB 1.096 (0.499–2.407) 0.819 Beta-block 0.939 (0.41–2.15) 0.882 Diagnosis and treatment EHRA score 1 1 (ref ) 2 1.477 (0.574–3.801) 0.419 3 1.184 (0.448–3.134) 0.733 CHA2DS2-VASc score ≥ 2 1.117 (0.58–2.153) 0.741 HAS-BLED score ≥ 3 0.599 (0.082–4.381) 0.614 Non-PV ablation 1.003 (0.516–1.947) 0.994 LAd, mm (≥ 40 vs. < 40) 1.966 (0.982–3.937) 0.056 LVEF, % (≥ 50% vs. < 50%) 1.126 (0.154–8.237) 0.907 Table 3 Adjusted hazard ratios of post-ablation persistent AF recurrence by gender Model adjustment Recurrence HR (95% CI) p value Unadjusted Gender 2.099 (1.087–4.053) 0.027 Model 1 Gender 2.205 (1.114–4.364) 0.023 Model 2 Gender 2.205 (1.081–4.497) 0.03 Model 3 Gender 2.371 (1.078–5.215) 0.032 Model 1: adjusted for age (≥ 65 years, < 65 years); model 2: adjusted for age, CAD, hypertension, stroke; model 3: adjusted for age, hypertension, CAD, stroke, LVEF (≥ 50%, < 50%), BMI (≥ 24 kg/m2, < 24 kg/m2), EHRA score, CHA2DS2-VASc score (≥ 2, < 2), HAS-BLED score (≥ 3, < 3) Abbreviations AF: Atrial fibrillation; CPVI: Circumferential pulmonary vein isolation; ECG: Electrocardiogram; HR: Hazard ratio; IQR: Interquartile range; LA: Left atrium; LVEF: Left heart ejection fraction; PV: Pulmonary vein; RFCA: Radiofrequency catheter ablation; TEE: Transesophageal echocardiography. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12872-022-03002-z. Additional file 1. Table S1. Baseline clinical characteristics of patients with atrial fbrillation. Additional file 2. Fig. S1. Kaplan-Meier survival curves for Freedom from AF/AT recurrence of all patients. Acknowledgements Not applicable. Author contributions HL and XZ designed the research study. ZW contributed to data collection. ZC and ZY contributed to follow-up, YZ and JG analyzed the data, YW designed and revised the manuscript. HL and XZ wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. Funding This work was supported by the Beijing Hospitals Authority Clinical Medicine Development of Special Funding Support (Grant No. XMLX202112). Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Declarations Ethics approval and consent to participate This study was approved by the ethics committee of the Beijing Anzhen Hospital (Approval NO: 2022042X) and was designed and performed in accordance with the Declaration of Helsinki. Informed consent was obtained from all participants. Consent for publication Not applicable. Competing interests All the authors have no conflicts of interest and had access to the data and wrote the manuscript. Author details 1 Beijing Anzhen Hospital, Capital Medical University, Beijing, People’s Republic of China. 2 Department of Cardiology, Beijing Anzhen Hospital, Capital Medical University, No. 2 Anzhen Road, Chaoyang District, Beijing 100029, People’s Republic of China. 3 Department of Radiation Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China. 4 Beijing Institute of Heart, Lung & Blood Vessel Disease, No. 2 Anzhen Road, Chaoyang District, Beijing 100029, People’s Republic of China. 5 The Key Laboratory of Remodeling‑Related Cardiovascular Diseases, Ministry of Education, Beijing, People’s Republic of China. Received: 15 July 2022 Accepted: 12 December 2022 References 1. 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Takigawa M, Takahashi A, Kuwahara T, Okubo K, Takahashi Y, Watari Y, Nakashima E, Nakajima J, Yamao K, Takagi K, Tanaka Y, Fujino T, Kimura S, Hikita H, Hirao K, Isobe M. Long-term outcome after catheter ablation of Page 7 of 7 paroxysmal atrial fibrillation: impact of different atrial fibrillation foci. Int J Cardiol. 2017;15(227):407–12. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Ready to submit your research ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations • maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions
https://openalex.org/W3131401159	http://qspace.qu.edu.qa/bitstream/10576/27448/1/materials-14-01086-v3.pdf	English	null	The Separation of Emulsified Water/Oil Mixtures through Adsorption on Plasma-Treated Polyethylene Powder	Materials	2,021	cc-by	11,798	Abdulkareem, A.; Popelka, A.; Sobolˇciak, P.; Tanvir, A.; Ouederni, M.; AlMaadeed, M.A.; Kasak, P.; Adham, S.; Krupa, I. The Separation of Emulsiﬁed Water/Oil Mixtures through Adsorption on Plasma-Treated Polyethylene Powder. Materials 2021, 14, 1086. https:// doi.org/10.3390/ma14051086 Abdulkareem, A.; Popelka, A.; Sobolˇciak, P.; Tanvir, A.; Ouederni, M.; AlMaadeed, M.A.; Kasak, P.; Adham, S.; Krupa, I. The Separation of Emulsiﬁed Water/Oil Mixtures through Adsorption on Plasma-Treated Polyethylene Powder. Materials 2021, 14, 1086. https:// doi.org/10.3390/ma14051086 Received: 24 December 2020 Accepted: 22 February 2021 Published: 26 February 2021 Keywords: oil/water emulsions; water treatment; adsorption; polyethylene; plasma treatment Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. The Separation of Emulsiﬁed Water/Oil Mixtures through Adsorption on Plasma-Treated Polyethylene Powder Abdulkareem 1, Anton Popelka 1, Patrik Sobolˇciak 1, Aisha Tanvir 1, Mabrouk Ouederni 2, m A. AlMaadeed 1 , Peter Kasak 1 , Samer Adham 3 and Igor Krupa 1,* Asma Abdulkareem 1, Anton Popelka 1, Patrik Sobolˇciak 1, Aisha Tanvir 1, Mabrouk Ouederni 2, Mariam A. AlMaadeed 1 , Peter Kasak 1 , Samer Adham 3 and Igor Krupa 1,* 1 Center for Advanced Materials, Qatar University, P.O. Box 2713 Doha, Qatar; asma.alkareem@qu.edu.qa (A.A.); anton.popelka@qu.edu.qa (A.P.); patrik@qu.edu.qa (P.S.); aisha.tanvir1991@gmail.com (A.T.); m.alali@qu.edu.qa (M.A.A.); peter.kasak@qu.edu.qa (P.K.) 2 QAPCO R&D–Qatar Petrochemical Company, P.O. Box 756 Doha, Qatar; MOuederni@qapco.com.qa 3 GWSC-ConocoPhillips, Qatar Science & Technology Park, Tech 2 Building, No.109, P.O. Box 24750 Doha, Qatar; Samer.Adham@conocophillips.com * C d i k @ d * Correspondence: igor.krupa@qu.edu.qa Abstract: This paper addresses the preparation and characterization of efﬁcient adsorbents for tertiary treatment (oil content below 100 ppm) of oil/water emulsions. Powdered low-density polyethylene (LDPE) was modiﬁed by radio-frequency plasma discharge and then used as a medium for the treatment of emulsiﬁed diesel oil/water mixtures in the concentration range from 75 ppm to 200 ppm. Plasma treatment signiﬁcantly increased the wettability of the LDPE powder, which resulted in enhanced sorption capability of the oil component from emulsions in comparison to untreated powder. Emulsions formed from distilled water and commercial diesel oil (DO) with concentrations below 200 ppm were used as a model of oily polluted water. The emulsions were prepared using ultrasonication without surfactant. The droplet size was directly proportional to sonication time and ranged from 135 nm to 185 nm. A sonication time of 20 min was found to be sufﬁcient to prepare stable emulsions with an average droplet size of approximately 150 nm. The sorption tests were realized in a batch system. The effect of contact time and initial oil concentrations were studied under standard atmospheric conditions at a stirring speed of 340 rpm with an adsorbent particle size of 500 microns. The efﬁciency of the plasma-treated LDPE powder in oil removal was found to be dependent on the initial oil concentration. It decreased from 96.7% to 79.5% as the initial oil concentration increased from 75 ppm to 200 ppm. The amount of adsorbed oil increased with increasing contact time. The fastest adsorption was observed during the ﬁrst 30 min of treatment. The adsorption kinetics for emulsiﬁed oils onto sorbent followed a pseudo-second-order kinetic model. materials materials Abdulkareem, A.; Popelka, A.; Sobolˇciak, P.; Tanvir, A.; Ouederni, M.; AlMaadeed, M.A.; Kasak, P.; Adham, S.; Krupa, I. The Separation of Emulsiﬁed Water/Oil Mixtures through Adsorption on Plasma-Treated Polyethylene Powder. Materials 2021, 14, 1086. https:// doi.org/10.3390/ma14051086 1. Introduction Wastewaters from various sources of the petroleum industry (gas, crude oil, shale gas extraction, and oil reﬁneries) represent the largest amounts of oily polluted water [1]. For example, the global production of processing wastewater was 202 billion barrels in 2014, and it is predicted to reach roughly 340 billion barrels in 2020 [2]. Wastewaters from the petroleum industry consist of both low molecular unsaturated and saturated hydro- carbons, and aromatic compounds such as benzene, toluene, xylene, and polyaromatic hydrocarbons [3]. These compounds have a mostly nonpolar character. On the other hand, wastewaters from the food industry and agriculture contain more polar natural oils and waxes [4]. The type of a treatment of oily polluted wastewaters depends on the oil concentration and can be categorized as primary, secondary, and tertiary treatment. Tertiary treatment is the puriﬁcation of wastewater having approximately 70–100 ppm oil components, and the required breakthrough concentration is around 5–10 ppm, de- pendently on national regulations. Oil removal by sorption through adsorption (often 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/materials Materials 2021, 14, 1086. https://doi.org/10.3390/ma14051086 Materials 2021, 14, 1086 2 of 15 2 of 15 accomplished by coalescence) using batch systems and percolating column conﬁgurations represents a fast and economically advantageous route for the tertiary treatment of oily polluted waters [1,5,6]. Because of ﬁne powders, which are useful in batch systems due to signiﬁcant pressure drop and are not suitable for deep-bed ﬁltration, the adsorbents applicable in deep-bed ﬁltration have mostly granular form [7,8]. pp p y g Commonly used sorbents can be prepared from suitable natural products (walnut shell, pecan, chitosan-rich structures) or synthetic polymers which have controlled geometry and surface characteristics. Polymeric materials seem to be the most promising sorbents in general because of their low cost, selectivity, and variability of their morphology. The surface of these materials can be additionally modiﬁed through various chemical and physical methods [9]. Plasma treatment represents the most powerful tool for the surface modiﬁcation of polymeric surfaces and is frequently used in many polymer-oriented industry applica- tions [10]. Plasma treatment represents a dry, clean, eco-friendly technique for the surface modiﬁcation of various materials (polymer, metal, wood, glass, etc.) [11]. 2.1. Materials Low-density polyethylene (LDPE) Lotrene FB3003 (MFI = 0.3 g/10 min, 190 ◦C, 2.16 kg; speciﬁc density = 0.92 g/cm3) in granular form supplied by Qatar Petrochemical Company (QAPCO, Doha, Qatar) was used as a raw material. LDPE granules were ground into powder form and sieved to obtain fractions of different sizes. A fraction with an average diameter (lateral dimension) of 0.5 mm was used for all experiments. Ethanol (Sigma Aldrich, Darmstadt, Germany), ultrapure water (Puriﬁcation System Direct Q3, Molsheim, France), and commercial diesel oil (DO) (petrol distribution company Woqod, Qatar) were used. DO is mostly composed of alkanes (C10–C32), as determined by gas chromatography. 1. Introduction The interactions of plasma-created species with a polymer surface can lead to different processes depending on the conditions used, such as the gas/gas mixture (Ar, N2, O2, CO2, NH3) and processing parameters (pressure, nominal power, treatment time, gas ﬂow rate) [12]. The introduc- tion of chemical functionalities can occur as a result of plasma oxidation, amination or nitration, while using gases without susceptibility for the formation of polymerizable intermediates after excitation. Moreover, free radical formation on a polymer surface can lead to surface activation, etching, ablation, or crosslinking processes [13]. Plasma treat- ment is thus responsible for changes in chemical composition accompanied by changes in topography/roughness. Moreover, it allows further interactions with other low- and high- molecular-weight species, and, therefore, enables modiﬁcation of surfaces by various com- pounds (grafting) with desirable functionalities. Since all these changes are realized only in the top surface layer, the original physical properties are unchanged. All these modiﬁca- tions are realized on the ﬁnal product, which is a very suitable technological route [14,15]. This paper is focused on the separation of oily components from emulsiﬁed wa- ter/diesel oil (DO) mixtures with oil contents of up to 200 ppm by plasma-treated low- density polyethylene (LDPE) powder in batch conﬁgurations. Plasma treatment was performed to improve the wettability of the LDPE sorbent by the emulsion, and, thus, to enhance its adsorption capacity. 2.6. Surface Area Measurements A BET surface area analyzer (Micromeritics-TriStar, Norcross (Atlanta), GA, USA) was employed to measure the speciﬁc surface area and pore size of the chosen grinded fraction of LDPE. The Brunauer-Emmet-Teller (BET) multipoint approach was employed to assess the surface area and pore distribution through nitrogen gas. The sample speciﬁc surface area was extrapolated at low temperatures of 70 ◦C from the amount of nitrogen (extremely small molecule) adsorbed onto the LDPE sample layer. 2.2. Preparation of Emulsions A mixture of 500 mL distilled water and 200 ppm DO was sonicated for 15 min at 40% amplitude using an ultrasonic sonicator (HIELSCHER UP400S, Berlin, Germany) device with a 22 mm titanium probe used as the homogenizer. Then, the stock solution was diluted with deionized water to prepare the required concentrations. 3 of 15 Materials 2021, 14, 1086 2.3. Plasma Treatment 2.4. Surface Wettability Analysis An OCA35 optical system (DataPhysics, Filderstadt, Germany) equipped with a CCD camera was employed to measure the wettability of the ﬂat LDPE surfaces (ﬁlms) after plasma treatment using static contact angle measurements via the sessile drop technique. Liquids with different surface tensions were tested to characterize the wettability of PE by an assessment of the surface free energy (γ) and its dispersive (γd) and polar (γp) components by using the Owens-Wendt-Rabel-Kaelble regression model [16]. 2.5. Surface Morphology/Topography Analysis The surface morphology of the untreated and treated LDPE (powder) samples was characterized with a ﬁeld emission scanning electron microscope (FEI-SEM, Nova Nano SEM 450, Hillsboro, OR, USA) facilitated by energy-dispersive X-ray spectroscopy (EDS) using secondary electron images at 3 kV and varying magniﬁcation. The specimens were sputter-coated with an approximately 2 nm layer of gold before taking SEM images to avoid the accumulation of electrons in the measured layer and to obtain SEM images with high resolution. g The surface topography of the LDPE powder samples was characterized by proﬁlom- etry (The Optical Surface Metrology System Leica DCM8, Mannheim, Germany). This system allows measuring the 3D surface topography of larger surface areas with no limits to the roughness. It contains ﬁve objectives with different magniﬁcations (5×, 10×, 20×, 50×, 100×), allowing for analysis of samples using different-size areas, and a highly sensi- tive detector (1.4 million pixel resolution) was used for obtaining confocal images. An EPI 100X-L objective (1360 × 1024 data points) was used to obtain the maximal detailed images from a surface area of 175.31 × 131.97 µm2. 2.3. Plasma Treatment Low-temperature plasma treatment of LDPE powder was performed using a Venus75- HF radio frequency (RF) plasma system (Plasma Etch Inc., Carson, CA, USA) in an air under vacuum. During plasma treatment, plasma-created reactive species were generated by means of an RF power supply operating at a typical frequency of 13.56 MHz. The chamber of the plasma system was evacuated to a pressure of approximately 0.2 Torr using a rotary vacuum pump prior to plasma application. Optimization of the treatment process was performed at various treatment times (10–180 s) to obtain the optimal wettability of the plasma-treated LDPE ﬁlm. The optimized plasma treatment was then applied to the treat the LDPE samples in powder form, which were placed in closed Petri dishes wrapped by parafﬁn ﬁlm during the plasma treatment in air, while the samples were turned over several times to ensure homogenous treatment for each side. 2.7. Chemical Composition Investigation FTIR was used to qualitatively evaluate changes in the chemical composition of LDPE untreated and plasma-treated surfaces. For this analysis, an FTIR spectrometer frontier (PerkinElmer, Waltham, MA, USA) ﬁtted with a ZnSe crystal was utilized, while the penetration depth of the infrared light was 1.66 µm. In addition, the spectral resolution and number of scans were set to 4 and 8, respectively. Qualitative information was obtained for the absorption of chemical groups in the middle infrared region (4000–500 cm−1). 4 of 15 Materials 2021, 14, 1086 X-ray photoelectron spectroscopy (XPS) was used for deeper characterization of the chemical composition changes induced by plasma treatment of LDPE powder. XPS spectra were captured using an Axis ultra DLD system (Kratos Analytical, UK) containing an Al Ka X-ray source. The sampling depth was in the range of 1–10 nm, allowing one to analyze only the top layer affected by plasma treatment (a few tens of nm). 2.9. Total Organic Carbon (TOC) Analysis TOC analysis was realized using a Formacs TOC/TN analyzer (Skalar Analyzer, Breda, The Netherlands). The samples were injected into a high-temperature combustion furnace where organic carbon (OC) was converted to carbon dioxide at 850 ◦C by catalytic oxidation (Pt catalyst). The formed CO2 was then dispersed into the carrier gas, and the concentration was measured by using a nondispersive infrared detector (NDIR). The total inorganic carbon (TIC) was determined by injecting the sample into a reactor containing acid (H3PO4) converting TIC into carbon dioxide. The concentration of the related CO2 was then determined by NDIR. Finally, TOC was calculated by subtracting TIC from TC. 2.8. Sorption of Oil from Emulsions In this study, the mass of the LDPE powder for all the experiments was arbitrarily chosen to be 3 g, and the volume of the tested emulsion was 40 mL. The mass of the powder was selected to respect the volume/mass ratio of the used vial. For example, 3 g of powder occupied approximately 2/3 of the total volume of the vial (45 mL). 3.1. Wettability of LDPE Prior to plasma treatment of the LDPE powders, optimization of the processing conditions was realized through plasma treatment of LDPE ﬁlms and wettability analysis. Plasma treatment time was in the range from 10 s to 180 s at a constant nominal power of 80 W. The contact angles and surface free energy for water, formamide, and ethylene glycol on the LDPE ﬁlms are shown in Figure 1a,b. The contact angles of the untreated LDPE surface achieved relatively high values, indicating hydrophobic character (high wettability). Plasma treatment was responsible for the decrease in contact angles with the increase in treatment time as a result of the formation of polar functionalities on the LDPE surface. After reaching 60 s of plasma treatment time, only slight changes in contact angles were observed. Therefore, this treatment time was also applied for plasma treatment of LDPE powder. VIEW 5 of 16 0 50 100 150 200 0 20 40 60 80 100 120 Contact angle (deg) Treatment time (s) Water Formamide Ethylene glycol 0 20 40 60 80 100 120 140 160 180 200 0 10 20 30 40 50 60 70 80 SEF Dispersive polar Surface free energy (m/m2) Treatment time (s) (a) (b) Figure 1. Dependence of (a) contact angle and (b) surface free energy (SFE) of selected liquids on plasma treated low- density polyethylene (LDPE) foil on treatment time. Figure 1. Dependence of (a) contact angle and (b) surface free energy (SFE) of selected liquids on plasma treated low-density polyethylene (LDPE) foil on treatment time. 0 20 40 60 80 100 120 140 160 180 200 0 10 20 30 40 50 60 70 80 SEF Dispersive polar Surface free energy (m/m2) Treatment time (s) (b) 0 50 100 150 200 0 20 40 60 80 100 120 Contact angle (deg) Treatment time (s) Water Formamide Ethylene glycol (a) Treatment time (s) (b) (a) (b) Figure 1. Dependence of (a) contact angle and (b) surface free energy (SFE) of selected liquids on plasma treated low density polyethylene (LDPE) foil on treatment time. Figure 1. Dependence of (a) contact angle and (b) surface free energy (SFE) of selected liquids on plasma treated low-density polyethylene (LDPE) foil on treatment time. 5 of 15 Materials 2021, 14, 1086 Figure 1. Depende density polyethylen The untreated and plasma-treated PE ﬁlms were also analyzed in terms of their surface wettability by water, DO, and 100 ppm emulsion. 3.1. Wettability of LDPE Illustration of the surface wettability of LDPE ﬁlms. 3.1. Wettability of LDPE The untreated LDPE surface showed hydrophobic and oleophilic characteristics; the water contact angle (WCA), oil contact angle (OCA) and emulsion contact angle (ECA) showed values of 95.3, 91.2, and 12.6◦, respectively (Figure 2). The plasma treatment resulted in a signiﬁcant improvement in wettability, while the WCA and ECA decreased to 57.8◦and 52.8◦, respectively. More- over, OCA achieved 5.2◦indicating very high oleophilicity. PE foil immersed into the DO emulsion shows no visible attachment of the oil droplets and DO droplets were freely moving (Figure 3A). On the other hand, PE ﬁlm immersed in DO formed a continuously covered oil layer with an average thickness of 1.4 ± 0.1 µm, as determined by proﬁlom- etry. These preliminary ﬁndings anticipate the applicability of plasma for improving oil/water separation. ce wettability by water, DO, and 100 ppm emulsion. The untreated LDPE surface howed hydrophobic and oleophilic characteristics; the water contact angle (WCA), oil ontact angle (OCA) and emulsion contact angle (ECA) showed values of 95.3, 91.2, and 2.6°, respectively (Figure 2). The plasma treatment resulted in a significant improvement n wettability, while the WCA and ECA decreased to 57.8° and 52.8°, respectively. More- ver, OCA achieved 5.2° indicating very high oleophilicity. PE foil immersed into the DO mulsion shows no visible attachment of the oil droplets and DO droplets were freely moving (Figure 3A). On the other hand, PE film immersed in DO formed a continuously overed oil layer with an average thickness of 1.4 ± 0.1 μm, as determined by profilometry. hese preliminary findings anticipate the applicability of plasma for improving oil/water eparation. Figure 2. Illustration of the surface wettability of LDPE films. Plasma treated LDPE Untreated LDPE DO OCA (θ = 12.6 o) Emulsion Pure water DO Emulsion Pure water ECA (θ = 91.2 o) WCA (θ = 95.3 o) OCA (θ = 5.2 o) ECA (θ = 52.8 o) WCA (θ = 57.8 o) OCA Oil Contact Angle ECA Emulsion Contact Angle WCA Water Contact Angle Figure 2. Illustration of the surface wettability of LDPE ﬁlms. Plasma treated LDPE Untreated LDPE DO OCA (θ = 12.6 o) Emulsion Pure water DO Emulsion Pure water ECA (θ = 91.2 o) WCA (θ = 95.3 o) OCA (θ = 5.2 o) ECA (θ = 52.8 o) WCA (θ = 57.8 o) Plasma treated LDPE igure 2. Illustration of the surface wettability of LDPE films. Figure 2. 3.2. Characterization of LDPE Powder Unlike LDPE pellets, which always have smooth surfaces as a consequence of the route of preparation (extrusion) (Figure 4A), LDPE powders, depending on the route of the preparation (mostly grinding; partly precipitation from a solution), can have very porous structures, as demonstrated by SEM and proﬁlometry analysis and shown in Figure 4B,C. The speciﬁc surface area of the powder was determined by BET analysis to be 44 ± 1 m2/g. A comparison of a simple estimation of the speciﬁc surface area of smooth pellets and porous grinded powders demonstrates enhancement of the speciﬁc surface of the ground powders. The pellet is approximated by a perfectly smooth sphere with diameter D = 500 µm, which corresponds to the size of the powder used in this study. The speciﬁc surface area of perfectly smooth spheres (Sa) can be calculated from Equation (1): sa = 6 ρD (1) sa = 6 ρD (1) D is the diameter of the uniform spheres, and ρ is the bulk density of the material. In our case, for D = 500 µm and ρ = 0.92 g·cm−3, and, therefore Sa = 0.013 m2/g, the experimentally determined value for the speciﬁc surface area of powder is 4.7 m2/g. This value is 362 times higher than the surface area of smooth spheres of the same size. This supports the statement that grinding neat pellets signiﬁcantly enhances the surface porosity of materials, and, thus, enhances the surface area of powders. 6 of 15 6 of 16 Materials 2021, 14, 1086 Materials 2021, 14, x FOR Figure 3. (A) The distribution of droplets of emulsion on the surface of treated PE foil; (B) Thin layer of DO deposited onto the surface of treated PE foil. The red line denotes the interface between the area covered by DO and the reference (non- immersed part of the foil); (C) Line profile for the surface of the treated PE foil. Figure 3. (A) The distribution of droplets of emulsion on the surface of treated PE foil; (B) Thin layer of DO deposited onto the surface of treated PE foil. The red line denotes the interface between the area covered by DO and the reference (non-immersed part of the foil); (C) Line proﬁle for the surface of the treated PE foil. Figure 3. 3.2. Characterization of LDPE Powder Nonetheless, this increase led to a reduction in the intensity of the C 1s peak to 90.2%. This effect is a result of the loss of some carbons throughout etching, radicalization, and substitution with groups containing oxygen [17]. Furthermore, as confirmed by Arpagaus et al. [18], the increase in peak intensity for powder LDPE is proof of an effective and homogeneous treatment, which is due to close contact between the powder surface and the plasma-created species [19]. (B) 8 of 16 (A) 21, 14, x FOR PEER REVIEW (A) (B) (C) Figure 4. SEM micrograph of a common as-produced LDPE pellet (A) SEM micrograph; (B) and profilometry image (C of LDPE powder prepared by grinding. Figure 4. SEM micrograph of a common as-produced LDPE pellet (A) SEM micrograph; (B) and proﬁlometry image (C) of LDPE powder prepared by grinding. in Table 1. All samples were investigated for the C 1s, O 1s, and N 1s spectra. The highest proportion in the XPS spectrum of untreated LDPE samples was reflected by the C 1s peak at ~280 eV. As shown in Table 1, the spectrum of untreated LDPE powder revealed high carbon atomic concentrations (at.%) equal to 99.0 at.%. In addition, negligible traces of oxygen and nitrogen-containing functional groups present in the spectra of untreated LDPE samples are likely attributed to processing additives or residual air within the plasma chamber [17]. The atomic concentrations of the O 1s and N 1s peaks for untreated LDPE are equal to 0.5 at.% and 0.5 at.%, respectively. Plasma treatment led to an increase in the intensity of the O 1s and N 1s peaks at binding energies of ~528 eV and ~400 eV, respectively, as illustrated in Figure 5. The O 1s and N 1s atomic percentages increased by 9.8% and 0.8% in powder LDPE post-treatment, respectively. Nonetheless, this increase led to a reduction in the intensity of the C 1s peak to 90.2%. This effect is a result of the loss of some carbons throughout etching, radicalization, and substitution with groups containing oxygen [17]. Furthermore, as confirmed by Arpagaus et al. [18], the increase in peak intensity for powder LDPE is proof of an effective and homogeneous treatment, which is due to close contact between the powder surface and the plasma-created species [19]. in Table 1. All samples were investigated for the C 1s, O 1s, and N 1s spectra. 3.2. Characterization of LDPE Powder The highe proportion in the XPS spectrum of untreated LDPE samples was reflected by the C 1s pe at ~280 eV. As shown in Table 1, the spectrum of untreated LDPE powder revealed hi carbon atomic concentrations (at.%) equal to 99.0 at.%. In addition, negligible traces oxygen and nitrogen-containing functional groups present in the spectra of untreat LDPE samples are likely attributed to processing additives or residual air within t plasma chamber [17]. The atomic concentrations of the O 1s and N 1s peaks for untreat LDPE are equal to 0.5 at.% and 0.5 at.%, respectively. Plasma treatment led to an increa in the intensity of the O 1s and N 1s peaks at binding energies of ~528 eV and ~400 e respectively as illustrated in Figure 5 The O 1s and N 1s atomic percentages increased (C) proo n the Figure 4. SEM micrograph of a common as-produced LDPE pellet (A) SEM micrograph; (B) and profilometry image (C of LDPE powder prepared by grinding. Figure 4. SEM micrograph of a common as-produced LDPE pellet (A) SEM micrograph; (B) and proﬁlometry image (C) of LDPE powder prepared by grinding. which is due to close contact between the powder surface and the plasma created species [19]. A comparison of a simple estimation of the specific surface area of smooth pellets and porous grinded powders demonstrates enhancement of the specific surface of the ground powders. The pellet is approximated by a perfectly smooth sphere with diamete D = 500 μm, which corresponds to the size of the powder used in this study. The specific surface area of perfectly smooth spheres (Sa) can be calculated from Equation (1): 𝑠𝑎= 6 𝜌𝐷a = 1, (1 D is the diameter of the uniform spheres, and ρ is the bulk density of the material. In our case, for D = 500 μm and ρ = 0.92 g·cm−3, and, therefore Sa = 0.013 m2/g, the experimen tally determined value for the specific surface area of powder is 4.7 m2/g. This value is 362 times higher than the surface area of smooth spheres of the same size. This supports the statement that grinding neat pellets significantly enhances the surface porosity of materi als, and, thus, enhances the surface area of powders. X-ray photoelectron spectroscopy (XPS) was employed to quantify the changes in the surface chemical composition of LDPE samples before and after plasma treatment in more detail. 3.2. Characterization of LDPE Powder (A) The distribution of droplets of emulsion on the surface of treated PE foil; (B) Thin layer of DO deposited onto the surface of treated PE foil. The red line denotes the interface between the area covered by DO and the reference (non- immersed part of the foil); (C) Line profile for the surface of the treated PE foil. Figure 3. (A) The distribution of droplets of emulsion on the surface of treated PE foil; (B) Thin layer of DO deposited onto the surface of treated PE foil. The red line denotes the interface between the area covered by DO and the reference (non-immersed part of the foil); (C) Line proﬁle for the surface of the treated PE foil. 7 of 15 7 of 1 Materials 2021, 14, 1086 Materials 2021, 14, x F (A) (B) (C) Figure 4. SEM micrograph of a common as-produced LDPE pellet (A) SEM micrograph; (B) and profilometry image (C) of LDPE powder prepared by grinding. A comparison of a simple estimation of the specific surface area of smooth pelle d i d d d d t t h t f th ifi f f t Figure 4. SEM micrograph of a common as-produced LDPE pellet (A) SEM micrograph; (B) and proﬁlometry image (C) of LDPE powder prepared by grinding. 2021, 14, x FOR PEER REVIEW 8 of 16 in Table 1. All samples were investigated for the C 1s, O 1s, and N 1s spectra. The highest proportion in the XPS spectrum of untreated LDPE samples was reflected by the C 1s peak at ~280 eV. As shown in Table 1, the spectrum of untreated LDPE powder revealed high carbon atomic concentrations (at.%) equal to 99.0 at.%. In addition, negligible traces of oxygen and nitrogen-containing functional groups present in the spectra of untreated LDPE samples are likely attributed to processing additives or residual air within the plasma chamber [17]. The atomic concentrations of the O 1s and N 1s peaks for untreated LDPE are equal to 0.5 at.% and 0.5 at.%, respectively. Plasma treatment led to an increase in the intensity of the O 1s and N 1s peaks at binding energies of ~528 eV and ~400 eV, respectively, as illustrated in Figure 5. The O 1s and N 1s atomic percentages increased by 9.8% and 0.8% in powder LDPE post-treatment, respectively. 3.3. Characterization of DO/Water Emulsions 3.3. Characterization of DO/Water Emulsions Emulsions are thermodynamically unstable systems due to their natural tendency to minimize interfacial interactions between chemically heterogeneous components [20,21]. From a practical point of view (for instance, because of a delay between emulsion prepa- ration, storage, and testing), it is important to know whether or not emulsions are stable over time. The evolution of droplet size over time is the key parameter for the stability of emulsion estimation since the instability is affected by changes in droplet size [22]. The stability of oil/water emulsions was inspected by Dynamic light scattering (DLS), directly determining the size of the droplets. The results are summarized in Figure 6. The droplet size remains fairly consistent over four days and is directly proportional to sonication time. A higher sonication time results in a smaller droplet size. To prepare a stable emulsion, twenty minutes of sonication was sufﬁcient. 9 of 16 remains fairly consistent over four days and is directly proportional to sonication . A higher sonication time results in a smaller droplet size. To prepare a stable emul- , twenty minutes of sonication was sufficient. 10 20 30 40 50 60 130 140 150 160 170 180 190 Droplet size (nm) Sonication Time (mins) Day 1 Day 2 Day 3 Day 4 ure 6. The dependence of the droplet size on the sonication time and the duration of emulsion age (until four days). Figure 6. The dependence of the droplet size on the sonication time and the duration of emulsion storage (until four days). re 6. The dependence of the droplet size on the sonication time and the duration of emulsion ge (until four days) Figure 6. The dependence of the droplet size on the sonication time and the duration of emulsion storage (until four days). 3.2. Characterization of LDPE Powder The XPS spectra measured for LDPE powder prior to and after treatment are shown in Figure 5. The quantification report for the atomic compositions of all samples is listed 1200 1000 800 600 400 200 0 Intensity (a.u) Binding Energy (eV) Untreated LDPE plasma treated LDPE N1s C1s O1s Figure 5. X-ray photoelectron spectroscopy (XPS) spectra for treated and untreated LDPE samples. Figure 5. X-ray photoelectron spectroscopy (XPS) spectra for treated and untreated LDPE samples. A comparison of a simple estimation of the specific and porous grinded powders demonstrates enhancemen ground powders. The pellet is approximated by a perfectl D = 500 μm, which corresponds to the size of the powder surface area of perfectly smooth spheres (Sa) can be calcul 𝑠𝑎= 6 𝜌𝐷a = 1, D is the diameter of the uniform spheres, and ρ is the our case, for D = 500 μm and ρ = 0.92 g·cm−3, and, therefore tally determined value for the specific surface area of pow times higher than the surface area of smooth spheres of t statement that grinding neat pellets significantly enhance als, and, thus, enhances the surface area of powders. X-ray photoelectron spectroscopy (XPS) was employe surface chemical composition of LDPE samples before and detail The XPS e t a ea u ed fo LDPE o de io t 1200 1000 800 600 400 200 0 Intensity (a.u) Binding Energy (eV) Untreated LDPE plasma treated LDPE N1s C1s O1s detail. The XPS spectra measured for LDPE powder prior to and after treatment are show in Figure 5. The quantification report for the atomic compositions of all samples is list e 5. X-ray photoelectron spectroscopy (XPS) spectra for treated and untreated LDPE samples. Figure 5. X-ray photoelectron spectroscopy (XPS) spectra for treated and untreated LDPE samples. 8 of 15 Materials 2021, 14, 1086 Table 1. XPS atomic composition summary for LDPE samples. Samples Element Atomic Conc. (at %) C 1s O 1s N 1s Untreated powder 99.0 0.5 0.5 Treated powder 90.2 9.58 0.8 3.3. Characterization of DO/Water Emulsions Table 1. XPS atomic composition summary for LDPE samples. Samples Element Atomic Conc. (at %) C 1s O 1s N 1s Untreated powder 99.0 0.5 0.5 Treated powder 90.2 9.58 0.8 3.3. Characterization of DO/Water Emulsions Table 1. XPS atomic composition summary for LDPE samples. age (until four days). 3.4. The Inﬂuence of Initial Oil Content The Influence of Initial Oil Content The initial oil portion in the emulsion can influence the kinetics of the adsorption, , therefore, this parameter has to be taken into account [23]. The influence of the initial concentration on the adsorption process under arbitrary conditions (3.0 g of adsor- t, 24 h, stirring) was investigated by varying the initial concentration from 75 ppm to ppm. The results are summarized in Table 2. It is evident that the quantity of oil ad- bed per unit weight of adsorbents qe increases with an increase in the initial oil content. the other hand, the oil removal efficiency decreased from 96.7% to 79.5% as the initial oncentration of the DO emulsion increased from 75 ppm to 200 ppm. This phenome- can be caused by the saturation of the available adsorption sites at higher oil concen- ons, which means that equilibrium between adsorbed oil and oil in the emulsion is hed at higher concentrations of oil in the emulsion. Similar results have been achieved The initial oil portion in the emulsion can inﬂuence the kinetics of the adsorption, and, therefore, this parameter has to be taken into account [23]. The inﬂuence of the initial DO concentration on the adsorption process under arbitrary conditions (3.0 g of adsorbent, 24 h, stirring) was investigated by varying the initial concentration from 75 ppm to 200 ppm. The results are summarized in Table 2. It is evident that the quantity of oil adsorbed per unit weight of adsorbents qe increases with an increase in the initial oil content. On the other hand, the oil removal efﬁciency decreased from 96.7% to 79.5% as the initial oil concentration of the DO emulsion increased from 75 ppm to 200 ppm. This phenomenon can be caused by the saturation of the available adsorption sites at higher oil concentrations, which means that equilibrium between adsorbed oil and oil in the emulsion is reached at higher concentrations of oil in the emulsion. Similar results have been achieved by Okie et al. 2011 [24] and Dirak et al. 2018 [25]. Materials 2021, 14, 1086 9 of 15 Table 2. Inﬂuence of initial oil concentration on the adsorption of emulsiﬁed diesel oil (DO) onto 3.0 g treated dosage at a contact time of 24 h. 3.0 g treated dosage at a contact time of 24 h. age (until four days). 3.4. The Inﬂuence of Initial Oil Content Initial Oil Concentration C0 (mg/L) Final Oil Concentration Ce (mg/L) Oil Removed, C0-Ce (mg/L) qe (mg/g) Removal Efﬁciency (%) 75 5.5 70 0.93 96.7 ± 0.8 100 11.0 76.5 1.19 93.5 ± 0.9 100 * 25.5 89 0.77 66.4 ± 3.0 150 22.3 128 1.70 86.7 ± 1.5 175 29.1 146 1.95 82.7 ± 1.8 200 34.5 166 2.21 79.5 ± 0.5 qe oil adsorbed per unit weight of adsorbent. * Removal efﬁciency of untreated LDPE powder determined under the same conditions. qe oil adsorbed per unit weight of adsorbent. * Removal efﬁciency of untreated LDPE powder determined under the same conditions. p ) Case II. 1 > RL > 0: The adsorption is favorable (a decrease in Gibbs free energy). 3.4.1. Adsorption Isotherms The value of the exponent n > 1 indicates a favorable adsorption of oil on the plasma-treated Case III. RL = 1 Characterization of a linear adsorption (unoccupied sites at the adsorbent are randomly occupied by adsorbate proportionally to their concentration, and only one reaction site is occupied by one species). VIEW 11 of 16 2 5 Case III. RL = 1 Characterization of a linear adsorption (unoccupied sites at the adsorbent are randomly occupied by adsorbate proportionally to their concentration, and only one reaction site is occupied by one species). h d bl IEW 11 of 16 2 5 only one reaction site is occu Case IV. RL = 0: The des 0.0 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 0.0 4 6 8 10 12 14 16 18 Ce/qe (mg/l) Ce (mg/L) Experimental data Linear fit 0.0 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 0.0 4 6 8 10 12 14 16 18 Ce/qe (mg/l) Ce (mg/L) Experimental data Linear fit Figure 7. Langmuir adsorption isotherm only one reaction site is occupied by one species). Case IV. RL = 0: The desorption process is irreversible. 0.0 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 0.0 4 6 8 10 12 14 16 18 Ce/qe (mg/l) Ce (mg/L) Experimental data Linear fit 0 5 10 15 20 25 30 35 40 0.0 0.5 1.0 1.5 2.0 2.5 Experimental data Non-linear fit qe (mg/g) Ce (mg/L) Figure 7. Langmuir adsorption isotherm nonlinear fit (right) and linear fit (left) Figure 7. Langmuir adsorption isotherm nonlinear ﬁt (right) and linear ﬁt (left). 0.0 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 0.0 4 6 8 10 12 14 16 18 Ce/qe (mg/l) Ce (mg/L) Experimental data Linear fit 0 5 10 15 20 25 30 35 40 0.0 0.5 1.0 1.5 2.0 2.5 Experimental data Non-linear fit qe (mg/g) Ce (mg/L) Figure 7. Langmuir adsorption isotherm nonlinear fit (right) and linear fit (left). y p y p Case IV. RL = 0: The desorption process is irreversible. ata 2.5 Experimental data 0 5 10 15 20 25 30 35 40 0.0 0.5 1.0 1.5 2.0 2.5 Experimental data Non-linear fit qe (mg/g) Ce (mg/L) 0 5 10 15 20 25 30 35 40 0.0 0.5 1.0 1.5 2.0 qe (mg/g) Ce (mg/L) nlinear fit (right) and linear fit (left). Ce (mg/L) e 7. 3.4.1. Adsorption Isotherms Langmu Figure 7 Langmuir adsorption isotherm nonlinear fit (right) and linear fit (left) Figure 7. Langmuir adsorption isotherm nonlinear ﬁt (right) and linear ﬁt (left). 0.0 1.5 2.0 2.5 3.0 3.5 -0.2 0.0 0.2 0.4 0.6 0.8 ln qe (mg/g) ln C (mg/L) Experimental data Linear fit 0 5 10 15 20 25 30 35 40 0.0 0.5 1.0 1.5 2.0 2.5 qe (mg/g) Ce (mg/L) Experimental data Non-linear fit 0.0 1.5 2.0 2.5 3.0 3.5 -0.2 0.0 0.2 0.4 0.6 0.8 ln qe (mg/g) ln Ce (mg/L) Experimental data Linear fit 0 5 10 15 20 25 30 35 40 0.0 0.5 1.0 1.5 2.0 2.5 qe (mg/g) Ce (mg/L) Experimental data Non-linear fit Figure 8. Freundlich adsorption isotherm nonlinear fit (right) and linear fit (left) Figure 8. Freundlich adsorption isotherm nonlinear ﬁt (right) and linear ﬁt (left). 0 2 0.0 0.2 0.4 0.6 0.8 ln qe (mg/g) Experimental data Linear fit 0.0 1.5 2.0 2.5 3.0 3.5 -0.2 0.0 0.2 0.4 0.6 0.8 ln qe (mg/g) ln C (mg/L) Experimental data Linear fit 0.0 0.5 1.0 1.5 2.0 2.5 qe (mg/g) Experimental data Non-linear fit 0 5 10 15 20 25 30 35 40 0.0 0.5 1.0 1.5 2.0 2.5 qe (mg/g) Ce (mg/L) Experimental data Non-linear fit 2.0 2.5 3.0 3.5 ln C (mg/L) 0 5 10 15 20 Ce (mg/L Figure 8. Freundlich adsorption isotherm nonlinear fit (right) and linear fit (left) Figure 8. Freundlich adsorption isotherm nonlinear ﬁt (right) and linear ﬁt (left). e ( g ) re 8. Freundlich adsorption isotherm nonlinear fit (right) and linear fit (left). p g Table 4. Langmuir and Freundlich isotherm parameters. The RL values are signiﬁcantly lower than that found for all tested initial concentra- tions (C0), indicating highly favorable adsorption of oil droplets on the adsorbent. e ( g ) . Freundlich adsorption isotherm nonlinear fit (right) and linear fit (left). able 4. Langmuir and Freundlich isotherm parameters. The RL values are signiﬁcantly lower than that found for all tested initial concentra- tions (C0), indicating highly favorable adsorption of oil droplets on the adsorbent. p Table 4. Langmuir and Freundlich isotherm parameters. 3.4.1. Adsorption Isotherms Adsorption isotherms contribute to the effective construction of adsorption systems, because they assess the maximum adsorption capacity that can be attained throughout the treatment process. The experimental results were evaluated using two of the most common isotherms, namely, the Langmuir [26–29] and Freundlich adsorption isotherms [30–32] (Table 3). The maximum adsorption capacity qm (mg/g) and adsorption (equilibrium) constants were determined. The ﬁtting data are summarized in Table 4 and Figures 7 and 8. The results revealed that the experimental data can be well ﬁtted by both the Langmuir and Freundlich isotherms with high correlation coefﬁcients (R2), which indicates mixed monolayer and multilayer adsorption. Table 3. Nonlinear and linear forms of the Langmuir and Freundlich isotherms. Equation Form Langmuir Isotherm Freundlich Isotherm Nonlinear qe = qm KLCe 1+KLCe qe = KFC 1 ne Linear Ce qe = 1 KLqm + Ce qm ln qe= ln KF + 1 n ln Ce Table 3. Nonlinear and linear forms of the Langmuir and Freundlich isotherms. Table 4. Langmuir and Freundlich isotherm parameters. Table 4. Langmuir and Freundlich isotherm parameters. Fitting Langmuir Isotherm Freundlich Isotherm qm (mg/g) KL (L/mg) 1 KLqm R2 KF (L/mg) n R2 Nonlinear 3.04 0.06 5.48 0.989 0.377 2.03 0.996 Linear 3.03 0.066 4.97 0.968 0.404 2.13 0.987 A useful parameter associated with the Langmuir isotherm is called the separation factor RL (Equation (2)): 1 RL = 1 1 + KLC0 (2) RL = 1 1 + KLC0 (2) (2) where RL indicates the shape of the isotherms, C0 (mg/L) is the initial concentration, and KL (L/mg) is the Langmuir constant related to the energy of adsorption from emulsions [32]: where RL indicates the shape of the isotherms, C0 (mg/L) is the initial concentration, and KL (L/mg) is the Langmuir constant related to the energy of adsorption from emulsions [32]: g g gy p RL corresponds to the adsorption processes according to the following criteria [32]: L co espo s o e so p o p ocesses cco g o e o o g c e [3 ] Case I. RL > 1: The adsorption is unfavorable (an increase in Gibbs free energy of adsorption). p Case II. 1 > RL > 0: The adsorption is favorable (a decrease in Gibbs free energy). 10 of 15 10 of 15 Materials 2021, 14, 1086 Case III. 3.4.1. Adsorption Isotherms RL = 1 Characterization of a linear adsorption (unoccupied sites at the adsorbent are randomly occupied by adsorbate proportionally to their concentration, and only one reaction site is occupied by one species). Case IV. RL = 0: The desorption process is irreversible. Materials 2021, 14, x FOR PEER REVIEW 11 of 16 0.0 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 0.0 4 6 8 10 12 14 16 18 Ce/qe (mg/l) Ce (mg/L) Experimental data Linear fit 0 5 10 15 20 25 30 35 40 0.0 0.5 1.0 1.5 2.0 2.5 Experimental data Non-linear fit qe (mg/g) Ce (mg/L) Figure 7. Langmuir adsorption isotherm nonlinear fit (right) and linear fit (left). 0.0 1.5 2.0 2.5 3.0 3.5 -0.2 0.0 0.2 0.4 0.6 0.8 ln qe (mg/g) ln Ce (mg/L) Experimental data Linear fit 0 5 10 15 20 25 30 35 40 0.0 0.5 1.0 1.5 2.0 2.5 qe (mg/g) Ce (mg/L) Experimental data Non-linear fit Figure 8. Freundlich adsorption isotherm nonlinear fit (right) and linear fit (left). Table 4. Langmuir and Freundlich isotherm parameters. Figure 7. Langmuir adsorption isotherm nonlinear ﬁt (right) and linear ﬁt (left). 0.0 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 0.0 4 6 8 10 12 14 16 18 Ce/qe (mg/l) Ce (mg/L) Experimental data Linear fit 0 5 10 15 20 25 30 35 40 0.0 0.5 1.0 1.5 2.0 2.5 Experimental data Non-linear fit qe (mg/g) Ce (mg/L) Figure 7. Langmuir adsorption isotherm nonlinear fit (right) and linear fit (left). 0.0 1.5 2.0 2.5 3.0 3.5 -0.2 0.0 0.2 0.4 0.6 0.8 ln qe (mg/g) ln Ce (mg/L) Experimental data Linear fit 0 5 10 15 20 25 30 35 40 0.0 0.5 1.0 1.5 2.0 2.5 qe (mg/g) Ce (mg/L) Experimental data Non-linear fit Figure 8. Freundlich adsorption isotherm nonlinear fit (right) and linear fit (left). Table 4. Langmuir and Freundlich isotherm parameters. Fitting Langmuir Isotherm Freundlich Isotherm qm ( / ) KL (L/ ) 𝟏 𝑲 R2 KF (L/mg) n R2 Figure 8. Freundlich adsorption isotherm nonlinear ﬁt (right) and linear ﬁt (left). The RL values are signiﬁcantly lower than that found for all tested initial concentra- tions (C0), indicating highly favorable adsorption of oil droplets on the adsorbent. Linear and nonlinear Freundlich isotherms are shown in Figure 8. The parameters (KF, 1/n) were determined from both linear and nonlinear ﬁtting of the experimental data. 3.4.2. Kinetics of Adsorption Kinetic models serve to estimate the duration of adsorption processes, and, thus, to estimate the time needed for the effective treatment of liquids. In batch systems, the solute concentration in the treated liquid gradually decreases with time until it reaches equilibrium with the adsorbed species. To determine the equilibrium time related to the maximum oil removal from emulsiﬁed oils [24], the amount of oil adsorbed onto the adsorbent was studied as a function of contact time in the range from 30 min to 1440 min using an initial oil concentration of 100 ppm and a treated adsorbent dose of 3.0 g. Equilibrium is achieved when the adsorption rate from the solution onto the surface of the sorbent corresponds to the rate of desorption from the sorbent to the solution [4]. As shown in Figure 9, the adsorption capacity (qt) increased over the whole duration of the sorption process; however, most of the oil was adsorbed within a short time (the ﬁrst 15 min) of the treatment. Thereafter, the oil removal efﬁciency reaches equilibrium when the rate of adsorption and desorption are equilibrated. This does not necessarily mean that the whole surface area is occupied and that the surface of the adsorbent does not need to be saturated with the oil. A high removal efﬁciency of 91.0% was achieved. The amount of adsorbed oil is directly proportional to the other parameters: the residual concentration of oil in the emulsion (Figure 10) and the percentage of removal (Figure 11). The ﬁrst parameter decreases over contact time, whereas the latter increases. 12 of 16 l oil concentration of 100 ppm and a treated adsorbent dose of 3.0 g. Equilibrium is ved when the adsorption rate from the solution onto the surface of the sorbent cor- nds to the rate of desorption from the sorbent to the solution [4]. As shown in Figure adsorption capacity (qt) increased over the whole duration of the sorption process; ver, most of the oil was adsorbed within a short time (the first 15 min) of the treat- Thereafter, the oil removal efficiency reaches equilibrium when the rate of adsorp- nd desorption are equilibrated. This does not necessarily mean that the whole sur- rea is occupied and that the surface of the adsorbent does not need to be saturated the oil. A high removal efficiency of 91.0% was achieved. 3.4.2. Kinetics of Adsorption The amount of adsorbed directly proportional to the other parameters: the residual concentration of oil in the sion (Figure 10) and the percentage of removal (Figure 11). The first parameter de- es over contact time, whereas the latter increases. 0.1 1 10 100 1000 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 qt (mg/g) log t (min) e 9. The dependence of the adsorption capacity (red squares) of DO on the contact time with ma-treated LDPE. Black line represents the nonlinear, pseudo-first-order (PFO) kinetic model. Figure 9. The dependence of the adsorption capacity (red squares) of DO on the contact time with plasma-treated LDPE. Black line represents the nonlinear, pseudo-ﬁrst-order (PFO) kinetic model. To describe the adsorption kinetics, two widely used models, namely, the pseudo- ﬁrst-order (PFO) and the pseudo-second-order (PSO) [33–35] kinetic models, were tested to ﬁt the experimental data. The common linear and nonlinear forms of those equations are shown in Table 5. The nonlinear form of PFO was used to keep the parameter qe as the adjustable parameter (Figure 9). It was found that PFO does not provide good agreement with the experimental data. The parameters of the PSO model, qe and k2, were determined from linear ﬁtting (Figure 12), and good agreement of the experimental data with the PSO model was demonstrated. All the parameters and constants determined from the PFO and PSO models are summarized in Table 6. The results conﬁrm that the adsorption kinetics for emulsiﬁed oils onto powder LDPE follow the PSO kinetic model. As reported by various authors, the PSO model better describes the sorption kinetics at lower initial concentrations [34–36] 0.1 1 10 100 1000 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 qt (mg/g) log t (min) e 9. The dependence of the adsorption capacity (red squares) of DO on the contact time with ma treated LDPE Black line represents the nonlinear pseudo first order (PFO) kinetic model Figure 9. The dependence of the adsorption capacity (red squares) of DO on the contact time with plasma-treated LDPE. Black line represents the nonlinear, pseudo-ﬁrst-order (PFO) kinetic model. 9. The dependence of the adsorption capacity (red squares) of DO on the contact time with t t d LDPE Bl k li t th li d fi t d (PFO) ki ti d l Figure 9. The dependence of the adsorption capacity (red squares) of DO on the contact time with plasma-treated LDPE. 3.4.1. Adsorption Isotherms Fitting Langmuir Isotherm Freundlich Isotherm q KL 𝟏 Fitting Langmuir Isotherm Freundlich Isotherm qm (mg/g) KL (L/mg) 𝟏 𝑲𝑳𝒒𝒎 R2 KF (L/mg) n R2 Nonlinear 3 04 0 06 5 48 0 989 0 377 2 03 0 996 Linear and nonlinear Freundlich isotherms are shown in Figure 8. The parameters (KF, 1/n) were determined from both linear and nonlinear ﬁtting of the experimental data. The value of the exponent n > 1 indicates a favorable adsorption of oil on the plasma-treated PE powder [23]. Fitting qm (mg/g) KL (L/mg) 𝟏 𝑲𝑳𝒒𝒎 R2 KF (L/mg) n R2 Nonlinear 3.04 0.06 5.48 0.989 0.377 2.03 0.996 Linear 3.03 0.066 4.97 0.968 0.404 2.13 0.987 3.4.2. Kinetics of Adsorption Nonlinear 3.04 0.06 5.48 0.989 0.377 2.03 0.996 Linear 3.03 0.066 4.97 0.968 0.404 2.13 0.987 3.4.2. Kinetics of Adsorption Kinetic models serve to estimate the duration of adsorption processes, and, thus, to estimate the time needed for the effective treatment of liquids. In batch systems, the solute qe is the amount of substance adsorbed at equilibrium per amount of adsorbent (mg/g), Ce is the equilibrium concentration (mg/L), qm is the saturation adsorption capacity (mg/g), and KL is the Langmuir equilibrium adsorption constant (L/mg). 1/n is the heterogeneity factor, n characterizes the intensity of the adsorption process, the relative distribution of the energy and the heterogeneity of the adsorbent reactive sites, and KF (L/mg) is the Freundlich adsorption constant. 11 of 15 11 of 15 Materials 2021, 14, 1086 3.4.2. Kinetics of Adsorption Black line represents the nonlinear, pseudo-ﬁrst-order (PFO) kinetic model. a treated LDPE. Black line represents the nonlinear, pseudo first order (PFO) kinetic model. To describe the adsorption kinetics, two widely used models, namely, the pseudo- ﬁrst-order (PFO) and the pseudo-second-order (PSO) [33–35] kinetic models, were tested to ﬁt the experimental data. The common linear and nonlinear forms of those equations are shown in Table 5. The nonlinear form of PFO was used to keep the parameter qe as the adjustable parameter (Figure 9). It was found that PFO does not provide good agreement with the experimental data. The parameters of the PSO model, qe and k2, were determined from linear ﬁtting (Figure 12), and good agreement of the experimental data with the PSO model was demonstrated. All the parameters and constants determined from the PFO and PSO models are summarized in Table 6. The results conﬁrm that the adsorption kinetics for emulsiﬁed oils onto powder LDPE follow the PSO kinetic model. As reported by various authors, the PSO model better describes the sorption kinetics at lower initial concentrations [34–36]. a treated LDPE. Black line represents the nonlinear, pseudo first order (PFO) kinetic model. To describe the adsorption kinetics, two widely used models, namely, the pseudo- ﬁrst-order (PFO) and the pseudo-second-order (PSO) [33–35] kinetic models, were tested to ﬁt the experimental data. The common linear and nonlinear forms of those equations are shown in Table 5. The nonlinear form of PFO was used to keep the parameter qe as the adjustable parameter (Figure 9). It was found that PFO does not provide good agreement with the experimental data. The parameters of the PSO model, qe and k2, were determined from linear ﬁtting (Figure 12), and good agreement of the experimental data with the PSO model was demonstrated. All the parameters and constants determined from the PFO and PSO models are summarized in Table 6. The results conﬁrm that the adsorption kinetics for emulsiﬁed oils onto powder LDPE follow the PSO kinetic model. As reported by various authors, the PSO model better describes the sorption kinetics at lower initial concentrations [34–36]. 12 of 15 Materials 2021, 14, 1086 0.01 0.1 1 10 100 1000 0 5 10 55 60 65 100 105 110 Ce (mg/L) log t (min) igure 10. The dependence of the residual concentration of DO on the contact time with the lasma-treated LDPE powder. Figure 10. 3.4.2. Kinetics of Adsorption The dependence of the residual concentration of DO on the contact time with the plasma- treated LDPE powder. 0.01 0.1 1 10 100 1000 0 5 10 55 60 65 100 105 Ce (mg/L) log t (min) igure 10. The dependence of the residual concentration of DO on the contact time with the lasma-treated LDPE powder. 0.01 0.1 1 10 100 1000 0 5 10 55 60 65 100 105 110 Ce (mg/L) log t (min) 0.01 0.1 1 10 100 1000 0 5 10 55 60 65 100 105 Ce (mg/L) log t (min) gure 10 The dependence of the residual concentration of DO o ure 10. The dependence of the residual concentration of DO on the contact time with the sma-treated LDPE powder. Figure 10. The dependence of the residual concentration of DO on the contact time with the plasma- treated LDPE powder. ma-treated LDPE powder. 0.01 0.1 1 10 100 1000 0 20 40 60 80 100 Percentage removal (%) log t (min) h d d f h f O l h h h l 0.01 0.1 1 10 100 1000 0 20 40 60 80 100 Percentage removal (%) log t (min) gure 11. The dependence of the percentage of DO removal on the contact time with the plasma- eated LDPE power. Figure 11. The dependence of the percentage of DO removal on the contact time with the plasma- treated LDPE power. 0 01 0 1 1 10 100 1000 0 20 40 60 80 100 Percentage removal (%) 0.01 0.1 1 10 100 1000 0 20 40 60 80 100 Percentage removal (%) log t (min) log t (min) ure 11. The dependence of the percentage of DO removal on the contact time with the plasma- ted LDPE power. Figure 11. The dependence of the percentage of DO removal on the contact time with the plasma- treated LDPE power. ated LDPE power. To describe the adsorption kinetics, two widely used models, namely, the pseudo- st-order (PFO) and the pseudo-second-order (PSO) [33–35] kinetic models, were tested fit the experimental data. The common linear and nonlinear forms of those equations e shown in Table 5. The nonlinear form of PFO was used to keep the parameter qe as the djustable parameter (Figure 9). 3.4.2. Kinetics of Adsorption k2 is the pseudo-second-order rate constant. U lik k hi h l h th di i i l t ti th t t k h able 5. Nonlinear and linear forms of the PFO and pseudo-second-order (PSO) kinetic models. Equation Form PFO Model PSO Model Nonlinear 𝑞𝑡 = 𝑞𝑒(1 −𝑒−𝑘1𝑡 ) 𝑞𝑡= 𝑞𝑒 2𝑘2 𝑡 1 + 𝑞𝑒𝑘2 𝑡 Li l ( ) 𝑙 𝑘 𝑡 1 + 𝑡 Table 6. Parameters of the PFO and PSO kinetic models. PFO Model PSO Model qe exp. (mg/g) qe ﬁt. (mg/g)s k1 (s−1) R2 qe ﬁt. (mg/g) k2 (g/min·mg) R2 1.23 1.22 0.0741 0.841 1.23 0.7092 0.989 Linear ln(𝑞𝑒 𝑞𝑡) 𝑙𝑛𝑞𝑒 𝑘1𝑡 𝑞𝑡 𝑞𝑒2 𝑘2 + 𝑞𝑒 qt is the amount of adsorbed species per mass of adsorbent (mg/g), k1 (min−1) is the seudo-first-order rate constant, qe is the amount of adsorbed species per mass of adsor- ent in equilibrium (mg/g), and t is time (min). k2 is the pseudo-second-order rate constant. nlike k1 which always has the dimension reciprocal to time the constant k2 may have qt is the amount of adsorbed species per mass of adsorbent (mg/g), k1 (min−1) is the pseudo-ﬁrst-order rate constant, qe is the amount of adsorbed species per mass of adsorbent in equilibrium (mg/g), and t is time (min). k2 is the pseudo-second-order rate constant. Unlike k1, which always has the dimension reciprocal to time, the constant k2 may have various dimensions (mg/g·min, g/g·min, mmol/g·min, etc.) 3.4.2. Kinetics of Adsorption It was found that PFO does not provide good agreement To describe the adsorption kinetics, two widely used models, namely, the pseudo- st-order (PFO) and the pseudo-second-order (PSO) [33–35] kinetic models, were tested fit the experimental data. The common linear and nonlinear forms of those equations e shown in Table 5. The nonlinear form of PFO was used to keep the parameter qe as the justable parameter (Figure 9). It was found that PFO does not provide good agreement th the experimental data. The parameters of the PSO model, qe and k2, were determined om linear fitting (Figure 12) and good agreement of the experimental data with the PSO Table 5. Nonlinear and linear forms of the PFO and pseudo-second-order (PSO) kinetic models. Equation Form PFO Model PSO Model Nonlinear qt = qe 1 −e−k1t qt = q2 e k2 t 1+qek2 t Linear ln(qe −qt) = lnqe −k1t t qt = 1 q2e k2 + t qe 13 of 15 Materials 2021, 14, 1086 1 10 100 1000 1 10 100 1000 log t/qt (min.g/mg) log t (min) Figure 12. The linear pseudo-second-order kinetic model. Red squares—experimental t/qt data. Figure 12. The linear pseudo-second-order kinetic model. Red squares—experimental t/qt data. gure 12. The linear pseudo-second-order kinetic model. Red squares—experimental t/qt data. Figure 12. The linear pseudo-second-order kinetic model. Red squares—experimental t/qt data. able 5. Nonlinear and linear forms of the PFO and pseudo- Table 6. Parameters of the PFO and PSO kinetic models. able 5. Nonlinear and linear forms of the PFO and pseudo-second-order (PSO) kinetic models. Equation Form PFO Model PSO Model Nonlinear 𝑞𝑡 = 𝑞𝑒(1 −𝑒−𝑘1𝑡 ) 𝑞𝑡= 𝑞𝑒 2𝑘2 𝑡 1 + 𝑞𝑒𝑘2 𝑡 Linear ln(𝑞𝑒−𝑞𝑡) = 𝑙𝑛𝑞𝑒−𝑘1𝑡 𝑡 𝑞𝑡 = 1 𝑞𝑒2 𝑘2 + 𝑡 𝑞𝑒 qt is the amount of adsorbed species per mass of adsorbent (mg/g), k1 (min−1) is the seudo-first-order rate constant qe is the amount of adsorbed species per mass of adsor- Table 6. Parameters of the PFO and PSO kinetic models. PFO Model PSO Model qe exp. (mg/g) qe ﬁt. (mg/g)s k1 (s−1) R2 qe ﬁt. (mg/g) k2 (g/min·mg) R2 1.23 1.22 0.0741 0.841 1.23 0.7092 0.989 qt is the amount of adsorbed species per mass of adsorbent (mg/g), k1 (min−1) is the pseudo-ﬁrst-order rate constant, qe is the amount of adsorbed species per mass of adsorbent in equilibrium (mg/g), and t is time (min). rious dimensions 4. Conclusions The fastest adsorption was observed during the ﬁrst 30 min of treatment. The adsorption kinetics for emulsiﬁed oils onto the sorbent followed the pseudo-second order kinetic model. 5. The adsorbed amount of oil increased with increasing contact time. The fastest adsorption was observed during the ﬁrst 30 min of treatment. The adsorption kinetics for emulsiﬁed oils onto the sorbent followed the pseudo-second order kinetic model. Author Contributions: Conceptualization, I.K., P.K., M.O. and M.A.A.; experimental, A.A.; resources, I.K.; data curation, A.A., A.P. and P.S.; writing—original draft preparation, P.S., A.T., A.P. and I.K.; writing—review and editing, I.K., P.K., S.A., P.S. and M.A.A.; project administration, I.K.; funding acquisition, I.K., M.A.A., M.O., P.K. and S.A. All authors have read and agreed to the published version of the manuscript. Funding: This work was made possible by a grant from the Qatar National Research Fund under its National Priorities Research Program (award number NPRP12S-0311-190299) and by ﬁnancial support from the ConocoPhillips Global Water Sustainability Center (GWSC). The paper’s content is solely the responsibility of the authors and does not necessarily represent the ofﬁcial views of the Qatar National Research Fund or ConocoPhillips. This research was also funded by Qatar University through Qatar University Collaborative Grant QUCGCAM- 20/21-3. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: Gas Processing Centrum at Qatar University is acknowledged for Total Or- ganic Carbon determination. SEM analysis was accomplished in the Central Laboratories Unit, Qatar University. Acknowledgments: Gas Processing Centrum at Qatar University is acknowledged for Total Or- ganic Carbon determination. SEM analysis was accomplished in the Central Laboratories Unit, Qatar University. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. rious dimensions 4. Conclusions able 6. Parameters of the PFO and PSO kinetic models. PFO Model PSO Model qe exp. (mg/g) qe fit. (mg/g)s k1 (s−1) R2 qe fit. (mg/g) k2 (g/min·mg) R2 1.23 1.22 0.0741 0.841 1.23 0.7092 0.989 LDPE powder prepared by grinding LDPE pellets and treating with radio-frequency plasma discharge was used as a polymer-based adsorption medium for the treatment of emulsiﬁed water/oil mixtures. Plasma treatment signiﬁcantly increased the wettability of the LDPE powder, which resulted in enhanced sorption efﬁciency. The sorption ability of untreated PE powder was also prechecked; however, due to a low oil removal efﬁciency, it was not investigated in detail, and, therefore, these results are not included in this paper. The ﬁndings can be summarized as follows: Conclusions LDPE powder prepared by grinding LDPE pellets and treating with radio-frequency asma discharge was used as a polymer-based adsorption medium for the treatment of mulsified water/oil mixtures. Plasma treatment significantly increased the wettability of LDPE d hi h l d i h d i ffi i Th i bili f 1. Emulsions formed from distilled water and commercial DO with concentrations below 200 ppm were used as a model of oily polluted water. The emulsions were prepared without emulsiﬁer, and emulsiﬁcation was ensured by ultrasonication. The long-term stability of emulsions was demonstrated by determining the evolution of oil droplet size over time. e LDPE powder, which resulted in enhanced sorption efficiency. The sorption ability of ntreated PE powder was also prechecked; however, due to a low oil removal efficiency, was not investigated in detail, and, therefore, these results are not included in this paper. h f d b d f ll p 2. It was found that the plasma-treated LDPE surface exhibited a highly hydrophilic character due to the incorporation of new polar functionalities on the surface, related to the change of the atomic composition indicated by the XPS method. he findings can be summarized as follows: g p y 3. The efﬁciency of the plasma-treated LDPE powder in oil removal was dependent on the initial oil concentration. It decreased from 96.7% to 79.5% as the initial oil concentration increased from 75 ppm to 200 ppm. 4. Freundlich isotherm better approximated the experimental points, which indicates mixed monolayer and multilayer adsorption. 14 of 15 14 of 15 Materials 2021, 14, 1086 5. The adsorbed amount of oil increased with increasing contact time. References 1. Pintor, A.M.A.; Vilar, V.J.P.; Botelho, C.M.S.; Boaventura, R.A.R. Oil and grease removal from wastewaters: Sorption treatment as an alternative to state-of-the-art technologies. A critical review. Chem. Eng. J. 2016, 297, 229–255. [CrossRef] 2. 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https://openalex.org/W3045874627	http://real.mtak.hu/115227/1/Bondarenko_Nanomat_2020-10-01499.pdf	English	null	Effects of Modified Magnetite Nanoparticles on Bacterial Cells and Enzyme Reactions	Nanomaterials	2,020	cc-by	12,237	Received: 29 June 2020; Accepted: 27 July 2020; Published: 30 July 2020 Abstract: Current paper presents biological eﬀects of magnetite nanoparticles (MNPs). Relations of MNP’ characteristics (zeta-potential and hydrodynamic diameters) with eﬀects on bacteria and their enzymatic reactions were the main focus. Photobacterium phosphoreum and bacterial enzymatic reactions were chosen as bioassays. Three types of MNPs were under study: bare Fe3O4, Fe3O4 modiﬁed with 3-aminopropyltriethoxysilane (Fe3O4/APTES), and humic acids (Fe3O4/HA). Eﬀects of the MNPs were studied at a low concentration range (< 2 mg/L) and attributed to availability and oxidative activity of Fe3+, high negative surface charge, and low hydrodynamic diameter of Fe3O4/HA, as well as higher Fe3+ content in suspensions of Fe3O4/HA. Low-concentration suspensions of bare Fe3O4 provided inhibitory eﬀects in both bacterial and enzymatic bioassays, whereas the MNPs with modiﬁed surface (Fe3O4/APTES and Fe3O4/HA) did not aﬀect the enzymatic activity. Under oxidative stress (i.e., in the solutions of model oxidizer, 1,4-benzoquinone), MNPs did not reveal antioxidant activity, moreover, Fe3O4/HA demonstrated additional inhibitory activity. The study contributes to the deeper understanding of a role of humic substances and silica in biogeochemical cycling of iron. Bioluminescence assays, cellular and enzymatic, can serve as convenient tools to evaluate bioavailability of Fe3+ in natural dispersions of iron-containing nanoparticles, e.g., magnetite, ferrihydrite, etc. Keywords: magnetite nanoparticles; humic acids-coated magnetite nanoparticles; silica-coated magnetite nanoparticles; zeta potential; hydrodynamic diameter; toxicity; bioluminescence; bacterial assay; enzymatic assay; oxidative stress; Photobacterium phosphoreum; NADH:FMN-oxidoreductase; luciferase Eﬀects of Modiﬁed Magnetite Nanoparticles on Bacterial Cells and Enzyme Reactions Lyubov S. Bondarenko 1, Ekaterina S. Kovel 2,3,* , Kamila A. Kydralieva 1, Gulzhian I. Dzhardimalieva 1,4 , Erzsébet Illés 5, Etelka Tombácz 5 , Arina G. Kicheeva 6 and Nadezhda S. Kudryasheva 3,6 Lyubov S. Bondarenko 1, Ekaterina S. Kovel 2,3,* , Kamila A. Kydralieva 1, Gulzhian I. Dzhardimalieva 1,4 , Erzsébet Illés 5, Etelka Tombácz 5 , Arina G. Kicheeva 6 and Nadezhda S. Kudryasheva 3,6 1 Moscow Aviation Institute (National Research University), 125993 Moscow, Russia; l.s.bondarenko92@gmail.com (L.S.B.); KydralievaKA@mai.ru (K.A.K.); dzhardim@icp.ac.ru (G.I.D.) 2 Institute of Physics SB RAS, FRC KSC SB RAS, 660036 Krasnoyarsk, Russia 3 Institute of Biophysics SB RAS, FRC KSC SB RAS, 660036 Krasnoyarsk, Russia; n-qdr@yandex.ru 4 Institute of Problems of Chemical Physics RAS, 142432 Chernogolovka, Moscow Region, Russia 5 University of Szeged, H-6720 Szeged, Hungary; Illes.Erzsebet@chem.u-szeged.hu (E.I.); E.Tombacz@chem.u-szeged.hu (E.T.) 6 g 6 Siberian Federal University, 660041 Krasnoyarsk, Russia; khyzylsyg@mail.ru * Correspondence: kkovel@yandex.ru; Tel.: +7-3912-494-242 nanomaterials nanomaterials nanomaterials 1. Introduction Magnetite nanoparticles (MNPs) have been widely used as adsorbents to extract ecotoxicants from waste and natural waters [1–3]. Thus, iron oxide nanomaterials, including magnetite (Fe3O4) can be found in engineered and natural aqueous environments due to their numerous applications. Hence, fate of these particles in aquatic systems is an issue of a considerable concern. However, given that bare MNPs are susceptible to air oxidation [4] and are easily aggregated in aqueous systems, the stabilization of the iron oxide particles is mandatory. Functionalization of MNP surfaces is usually Nanomaterials 2020, 10, 1499; doi:10.3390/nano10081499 www.mdpi.com/journal/nanomaterials 2 of 20 Nanomaterials 2020, 10, 1499 desired, and various molecules can be used as capping agents. Humic acids (HA) being natural high-molecular compound, have a high aﬃnity to Fe3O4 nanoparticles; the sorption of HA on the Fe3O4 nanoparticles enhances the stability of the nanodispersions by preventing their aggregation [5–8]. Previously [8,9], magnetite Fe3O4 nanoparticles coated with HA were used for the extraction of heavy metal ions from water. Under certain conditions, these insoluble magnetic materials get an advantage of being easily removed with magnetic ﬁeld. Numerous studies reported a successful functionalization of Fe3O4 nanoparticles with active molecules such as alkoxysilanes [10–14]. It was also shown that alkoxysilanes and MNPs are interconnected by strong covalent bonds. A functional molecule like alkoxysilane consists of at least two functional groups, where the ﬁrst group is applied for the attachment to the MNP surface, while the second group provides an adhesion point to the molecule bond [15]. The Fe3O4 nanoparticles grafted with SiO2 and alkoxysilanes are promising nanobiotechnological reagents due to their biocompatibility and hydrophilic properties. A huge variety of alkoxysilanes allows for the diﬀerent types of surface functionalization by introducing charges into the system. As a result, the functionalization prevents the nanoparticle’ aggregation in liquids and improves their chemical stability [16]. Various applications of magnetite-alkoxysilane nanoparticles are known, such as detoxifying agents in ecological control [17], for drug delivery systems in medicine [18], technical use as thermal insulators [19], for soil stabilization [20–22], and in signiﬁcant soil improvement [23]. It has been reported that SiO2-based nanoparticles could be used as fertilizers to enhance a seed germination and stimulate antioxidant system of crops [24]; they could also be used for pesticide delivery [25] and increasing the SiO2 entering into the terrestrial system [26]. A wide application range of MNPs encourages the deeper understanding of their biological activity. 1. Introduction Since modiﬁed MNPs are potential adsorbents, their eﬀects on water microorganisms are of special interest. Eﬀects of modiﬁed MNPs on aqueous microorganisms are currently under study; a series of agents, such as anchored SnO2 [27], TiO2 [28], chitosan [29], Ag [30], oleic acid [31], and glycine [32,33] were previously used to modify an MNP surface. Oleic acid and glycine modiﬁcations showed lower cell toxicity as compared to bare nanoparticles, whereas TiO2 revealed a higher antibacterial activity. In this study, we chose luminous marine bacterium as a model microorganism to evaluate and compare biological eﬀects of three types of magnetite nanoparticles: bare MNPs, modiﬁed by humic acids, and silica (3-aminopropyltriethoxysilane, APTES), i.e., Fe3O4, Fe3O4/HA, and Fe3O4/APTES, respectively. The bioluminescence bacteria-based assay is a traditional toxicity analysis; it has been applied for more than 50 years for toxicity monitoring due to its high sensitivity [34–39]. This bioassay uses luminescence intensity as a physiological test parameter. The registration of bioluminescence provides a convenient approach with high rates, low costs, and convenience of the bioassay procedure. The bioassay is widely applied to monitor a general toxicity of complex media, as well as a toxicity of individual chemical species [34–38] and radiation [40,41]. To evaluate molecular mechanisms of possible biological eﬀects of the MNPs, enzymatic bioassays can be applied. The bioluminescence enzymatic assay is a relatively new direction in a toxicology practice [42–44]. The conventional enzymatic bioluminescent assay is based on the bacterial bioluminescent enzyme system, which involves two coupled enzymatic reactions: NADH + FMN NADH:FMN−oxidoreductase −−−−−−−−−−−−−−−−−−−−−→FMN · H−+ NAD+ (R1) FMN · H−+ RCHO + O2 luci ferase −−−−−−−→FMN + RCOO−+ H2O + hv (R2) (R1) FMN · H−+ RCHO + O2 luci ferase −−−−−−−→FMN + RCOO−+ H2O + hv (R2) (R2) Similar to cellular bioassay, the enzymatic assays can assess a general toxicity in test samples. This toxicity type integrates all interactions of toxic compounds with the bioluminescent assay system: redox processes, polar and nonpolar binding, etc. Moreover, the enzymatic bioassay is speciﬁc to oxidizers due to its additional kinetic parameter, induction period, which depends on redox potentials of toxicants in solutions [44]. Therefore, the enzymatic bioassay can be additionally applied to monitor 3 of 20 Nanomaterials 2020, 10, 1499 a toxicity of oxidative type, which is attributed to the redox properties of the toxic compounds only. 1. Introduction Due to such features, the enzymatic bioassay can be used to reveal the ability of MNPs to interfere the redox reactions, as well as stimulate/inhibit biochemical processes as a result of surface hydrophobic/hydrophilic interactions. Previously [45,46], we used the bioluminescent enzymatic assay system to evaluate the toxicities of general and oxidative types in solutions of organic and inorganic oxidizers (quinones and polyvalent metals, respectively). Changes in general toxicity and oxidative toxicity of aqueous media under exposure to humic substances were studied previously in [47–49]. Later [41,50–52], the toxicity and antioxidant activity of a series of fullerenols (i.e., carbon nanostructures, water-soluble derivatives of fullerenes, perspective pharmaceutical agents) were evaluated and compared. Eﬀects of MNPs and MNPs with modiﬁed surface on bacterial enzyme reactions have not been studied yet. Current paper compares the biological eﬀects of MNPs with their zeta potentials and hydrodynamic diameters. Chemical precipitation method in situ and sol–gel synthesis were used to produce bare and modiﬁed MNPs. The MNPs were characterized by XRD, SEM, and DLS analysis. Toxicity eﬀects of MNPs were evaluated and compared using a model cellular bioassay–luminous marine bacteria. Ability of MNPs to aﬀect the biochemical processes was studied using a bioluminescence assay based on enzyme reactions of the luminous bacteria. A vital role of a ligand type (HA or APTES) in the eﬀects of MNPs on the enzymatic activity was under consideration. 2. Materials and Methods 2.1. Preparations of Fe3O4 MNPs and Humic Acids- and Amino-Silica Functionalized Fe3O4 MNPs 2.1.2. Preparation of the Silica-Coated Fe3O4/APTES Sample To prepare the aminosilica-functionalized MNPs the surface modiﬁcation of Fe3O4 was performed using 3-aminopropyltriethoxysilane (APTES, (Sigma-Aldrich Chemie GmbH, Steinheim, Germany)) as a silylation agent according to [54]. Brieﬂy, 3.21 g of Fe3O4 was dispersed in 150 mL of ethanol/water (volume ratio, 1:1) solution. Then, 13.6 g of APTES was added into the solution under argon atmosphere, at 40 ◦C for 2 h; molar ratio of APTES to the Fe3O4 was applied as 4:1. After that, the solution was cooled down to a room temperature. The prepared Fe3O4/APTES MNPs were collected with a magnet (Nd, 0.3 T) and rinsed three times with ethanol and deionized water. Finally, the Fe3O4/APTES sample was dried in vacuum at 70 ◦C for 2 h. 2.1.1. Preparation of the Fe3O4 MNPs The bare Fe3O4 MNPs were prepared by the coprecipitation of Fe (II, III) salts in base solution as described in detail in [53]. Brieﬂy, 7.56 g of FeCl3·6H2O (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and 2.78 g of FeCl2·4H2O (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) were dissolved in 70 mL H2O, then added to 40 mL of 25% solution of ammonium hydroxide (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) at 50 ◦C under argon ﬂow with vigorous stirring at 600 rpm. The formed Fe3O4 nanoparticles were washed ﬁve times with Millipore water and ethanol to eliminate the impurities from synthesis followed by drying at 70 ◦C in vacuum. 2.1.2. Preparation of the Silica-Coated Fe3O4/APTES Sample 2.2. Characterization of the MNPs The phase composition and particle size of the MPNs samples were determined by X-ray diﬀraction analysis (XRD) in Bragg-Brentano geometry using a Philips X-Pert Diﬀractometer (Cr-Kα radiation, λ = 2.29106 Å (Philips Analytical, Eindhoven, The Netherlands)). The full width at a half maximum (FWHM) of the all reﬂections was used for particle size determination with the Scherrer equation. In order to quantify oxidation progress, the (440) reﬂection was ﬁtted with ﬁve diﬀerent functions in Origin 2019 Pro (OriginLab Corporation, Northampton, MA, USA). The morphology of the nanomaterials was investigated by scanning electron microscopy (SEM) using a Zeiss LEO SUPRA 25 microscope (ZEISS, Jena, Germany). The average particle diameter of the synthesized MNPs was determined by measuring the diameters of more than 100 particles with ImageJ software ± standard deviation. The data were ﬁtted with Lognormal functions (95% conﬁdence interval) using Origin 2019 Pro (Table S1, Supplementary Materials). Dynamic light scattering (DLS) measurements were conducted with a NanoZS apparatus (Malvern Panalytical Ltd., Malvern, UK) at a wavelength of 633 nm with a solid-state He–Ne laser at a scattering angle of 173◦at 25 ◦C. The intensity, volume, and number average diameters of MNP samples were calculated with the Zetasizer Nano 7.11 software (Brookhaven Instruments, Holtsville, NY, USA) utilizing an algorithm based on the Mie theory, which transforms time-varying intensities to particle diameters [56]. For the DLS analysis, each sample was diluted to approximately 0.1 g/L. Since the colloidal stability of the samples varies with the solution conditions, the particles may aggregate; all were measured at a given kinetic state achieved by 10 s of ultrasonication followed by 100 s of standstill. The average values of the hydrodynamic diameter were calculated from 3rd order cumulant ﬁts of the correlation functions. The experiments were carried out in a disposable zeta cell (DTS 1070). The range of pH was between ~3 and ~10. The values of pH before and after the study were measured, subsequently; the value of pH after measurement by the method of dynamic light scattering was used. The experiments were performed at constant ionic strengths (0.01 M) set by NaCl. Data of zeta potential from pictures (dots) was ﬁtted with Boltzmann functions (lines) and 95% conﬁdence band in Origin 2019 Pro (Table S1, Supplementary Materials). 2.3. Bioluminescence Assay Systems and Experimental Data Processing 2.3. Bioluminescence Assay Systems and Experimental Data Processing Eﬀects of the MNPs on microbiological and biochemical processes were evaluated using model bioluminescence assay systems, both cellular and enzymatic, i.e., luminous marine bacteria and a system of coupled enzymatic reactions of the marine bacteria, respectively. The bacterial assay, i.e., Microbiosensor 677F, was based on the lyophilized luminous bacteria Photobacterium phosphoreum from the collection of the Institute of Biophysics SB RAS (CCIBSO 863), strain 667F IBSO. The enzyme preparation was based on the system of coupled enzyme reactions catalyzed by NADH:FMN-oxidoreductase from Vibrio ﬁscheri (0.15 a.u.) and luciferase from Photobacterium leiognathi (0.5 mg/mL) [57]. The enzyme reactions (1) and (2) are presented in Introduction. All biological preparations were produced at the Institute of Biophysics SB RAS, Krasnoyarsk, Russia. The following chemicals were used: NADH from MP Biomedicals, Santa Ana, CA, USA; FMN and tetradecanal from Sigma-Aldrich Chemie GmbH, St. Louis, MO, USA; 1,4-benzoquinone (Bq) from ACROS ORGANICS, NJ, USA; and sodium chloride (NaCl) from Khimreactivsnab, Barnaul, Russia. To construct the enzymatic assay system, we used 0.1 mg mL−1 enzyme preparation, 4 × 10−4 M NADH, 5.4 × 10−4 M FMN, and 0.0025% tetradecanal solutions. The enzymatic assay was performed in 0.05 M phosphate buﬀer, pH 6.8, at 20 ◦C. The bacterial assay, i.e., Microbiosensor 677F, was based on the lyophilized luminous bacteria Photobacterium phosphoreum from the collection of the Institute of Biophysics SB RAS (CCIBSO 863), strain 667F IBSO. The enzyme preparation was based on the system of coupled enzyme reactions catalyzed by NADH:FMN-oxidoreductase from Vibrio ﬁscheri (0.15 a.u.) and luciferase from Photobacterium leiognathi (0.5 mg/mL) [57]. The enzyme reactions (1) and (2) are presented in Introduction. All biological preparations were produced at the Institute of Biophysics SB RAS, Krasnoyarsk, Russia. g p p p p y y The following chemicals were used: NADH from MP Biomedicals, Santa Ana, CA, USA; FMN and tetradecanal from Sigma-Aldrich Chemie GmbH, St. Louis, MO, USA; 1,4-benzoquinone (Bq) from ACROS ORGANICS, NJ, USA; and sodium chloride (NaCl) from Khimreactivsnab, Barnaul, Russia. To construct the enzymatic assay system, we used 0.1 mg mL−1 enzyme preparation, 4 × 10−4 M NADH, 5.4 × 10−4 M FMN, and 0.0025% tetradecanal solutions. The enzymatic assay was performed in 0.05 M phosphate buﬀer, pH 6.8, at 20 ◦C. 2.1.3. Preparation of the Humic Acids-Coated Fe3O4/HA Sample The Fe3O4/HA sample was prepared by in situ method, which consists of the nucleation and growth of magnetite nanoparticles into humic acids matrix. The sample of HA was prepared from sodium humate (Powhumus (Humintech GmbH, Grevenbroich, Germany)). The low H/C atomic ratio (0.85) suggests high content of aromatic structures in the HA preparation. The total acidity of the sample was 5.3 mmol/g of acidic COOH and OH-groups, weight-average molecular weight (Mw) was 9.9 kD. The MNPs modiﬁed with HA (as Fe3O4/HA ratio 80:20 wt %) were prepared by the procedure described in [53,55]. 4 of 20 Nanomaterials 2020, 10, 1499 2.2. Characterization of the MNPs Data of hydrodynamic size were ﬁtted with Lognormal, Extreme, and Boltzmann functions (lines) for Fe3O4, Fe3O4/APTES, and Fe3O4/HA, respectively, and 95% conﬁdence interval in Origin 2019 Pro (Table S1, Supplementary Materials). 2.3. Bioluminescence Assay Systems and Experimental Data Processing Preparation of the MNPs suspensions for the bioluminescence analyses: Solid samples were ground in a mortar and dissolved in 3% aqueous NaCl solution or 0.05 M potassium phosphate buﬀer for bacterial or enzymatic assays, respectively. To obtain homogeneous suspensions, the 1000 mg/L stock suspensions were exposed to the ultrasound using an Elmasonic EASY 10 bath (Elma Schmidbauer 5 of 20 Nanomaterials 2020, 10, 1499 GmbH, Singen, Baden-Wurttemberg, Germany) for 10 min. Analyzed MNPs concentrations (5–10−13 mg/L) were prepared from the stock suspensions, with additional 5 s ultrasonic treatment at every dissolution stage. Before the bioluminescence measurements, the dispersions were additionally treated in the ultrasonic bath for 5–10 s. To evaluate the eﬀect of optic ﬁlter on the MNPs’ suspensions, absorption spectra of the suspensions were recorded using UVIKON 943 Double Beam UV/VIS Spectrophotometer (Kontron Instruments, Milan, Italy). The suspensions of optical density higher than 0.1 (at 490 nm) were excluded, hence, the eﬀect of “optic ﬁlter” [58] did not skew the results the bioluminescence measurements. Measurements of bioluminescence intensity were carried out with bioluminometer Luminoskan Ascent (Thermo Electron Corporation, San Diego, CA, USA). To assess the toxic/inhibitory eﬀect of MNPs on the bacterial and enzymatic assays, the relative bioluminescence intensity, Irel, was calculated as: Irel = IMNP/Icontr (1) (1) where Icontr and IMNP are maximal bioluminescence intensities in the absence and presence of the MNPs, respectively. The experimental error for Irel did not exceed 8% in the bacterial assay and 12% in the enzymatic assay. The data for Irel processing were obtained with ﬁve samplings from all control and MNP solutions. The dependence of Irel on MNP concentrations was studied. To characterize toxic eﬀects of the MNPs on the bacteria and enzyme reactions, their eﬀective concentrations that inhibited luminescence intensity by 20% (Irel = 0.8), EC-20, were determined and compared. Additionally, the eﬀects of the MNPs on the enzyme system were studied under conditions of an oxidative stress. An organic oxidizer, 1,4-benzoquinone (Bq), was chosen for these experiments to model conditions of the oxidative stress. The eﬀective concentration of Bq that inhibited the bioluminescence intensity by 50% (8 × 10−6 M), was applied in all experiments. 2.3. Bioluminescence Assay Systems and Experimental Data Processing y y pp p To study and compare the eﬀects of the MNPs under conditions of the oxidative stress, the relative bioluminescence intensity, IrelBq, was calculated as: Irel Bq = IMNP+Bq/IBq (2) (2) where, IMNP+Bq and IBq are maximal bioluminescence intensities in Bq solutions in the presence and absence of MNPs, respectively (see Figure 1). To characterize the MNP eﬃciency to suppress the enzymatic activity under the oxidative stress, the eﬀective MNP concentrations that inhibited bioluminescence intensity by 50% (IrelBq = 0.5), EC-50, were determined and compared. Additionally, the speciﬁc eﬀects of MNPs on redox processes in the enzymatic assay system under conditions of the oxidative stress were studied, i.e., the bioluminescence induction periods (T0.5)Bq in Bq solutions were measured (Figure 1). The TrelBq values were calculated as: Trel Bq = (T0.5)Bq/(T0.5)Bq+MNP (3) (3) re (T0.5)Bq and (T0.5)Bq+MNP are bioluminescence induction periods in oxidizer (Bq) solutions in the ence and presence of MNPs, respectively (Figure 1). l l The IrelBq and TrelBq values were plotted vs. concentrations of the nanoparticles. l l The IrelBq and TrelBq values were plotted vs. concentrations of the nanopa l l The IrelBq and TrelBq values were plotted vs. concentrations of the nanoparticles. l l Values of IrelBq < 1 or TrelBq < 1 revealed an increase in inhibition eﬀects of the oxidizer solutions under the exposure to the MNPs. Values of IrelBq ≈1 or TrelBq ≈1 revealed the absence of the eﬀect. 6 of 20 f 20 Nanomaterials 2020, 10, 1499 nomaterials 2020, 10, x FOR PEE igure 1. Bioluminescence kinetics in solutions of model oxidizer 1,4-benzoquinone (Bq) and magnetite nanoparticles (MNPs). Enzymatic assay. Figure 1. Bioluminescence kinetics in solutions of model oxidizer 1,4-benzoquinone (Bq) and magnetite nanoparticles (MNPs). Enzymatic assay. Nanomaterials 2020, 10, x FOR PEER REVIEW 6 of 20 gure 1. Bioluminescence kinetics in solutions of model oxidizer 1,4-benzoquinone (Bq) and agnetite nanoparticles (MNPs). Enzymatic assay. Figure 1. Bioluminescence kinetics in solutions of model oxidizer 1,4-benzoquinone (Bq) and magnetite nanoparticles (MNPs). Enzymatic assay. esults and Discussion Experimental error for IrelBq and TrelBq did not exceed 12%. The data for IrelBq and TrelBq processin were obtained in two experiments with ﬁve samplings from all control and nanoparticle solutions. . Microstructure of MNPs The crystalline structures of the nanoparticles were identified with XRD analysis (Figure 2). 2.3. Bioluminescence Assay Systems and Experimental Data Processing The RD patterns are similar for all samples and can be interpreted as a face centered cubic (fcc) lattice Statistical processing of the biotesting results was carried out; p-values were calculated with RStudio using ANOVA and presented in Supplementary Materials, Tables S2–S5. The p-values were assessed by Student’s t-test of two independent sample distributions. Figure 1. Bioluminescence kinetics in solutions of model oxidizer 1,4-benzoquinone (Bq) and magnetite nanoparticles (MNPs). Enzymatic assay. p th the parameters of 8.383(2) l i l (T bl 1) 3. Results and Discussion 3. Results and Discussion mples, respectively (Table 1). 3.1. Microstructure of MNPs 3.1. Microstructure of MNPs The crystalline structures of the nanoparticles were identiﬁed with XRD analysis (Figure 2). The XRD patterns are similar for all samples and can be interpreted as a face-centered cubic (fcc) lattice with the parameters of 8.383(2), 8.372(1), and 8.382(6) Å for the Fe3O4, Fe3O4/APTES, and Fe3O4/HA samples, respectively (Table 1). f The crystalline structures of the nanoparticles were identified with XRD analysis (Figure 2). The XRD patterns are similar for all samples and can be interpreted as a face-centered cubic (fcc) lattice with the parameters of 8.383(2), 8.372(1), and 8.382(6) Å for the Fe3O4, Fe3O4/APTES, and Fe3O4/HA samples, respectively (Table 1). Figure 2. XRD patterns of MNPs samples. The lattice parameters determined for all samples formulated in this study are smaller than those ported for magnetite 8.396–8.400 Å (ICDD–PDF 19–629), but larger than those for maghemite 8.33– 34 Å (ICDD–PDF 39–1346). A plausible explanation of this phenomenon could be the process of artial oxidation of Fe2+ during drying and storage resulting in the nonstoichiometric Fe3-δO4 rmation where δ can range from zero (stoichiometric magnetite) to 1/3 (completely oxidized) [59] Figure 2. XRD patterns of MNPs samples. Figure 2. XRD patterns of MNPs samples. lausible explanation of this phenomeno drying and storage resulting in the Figure 2. XRD patterns of MNPs samples. Figure 2. XRD patterns of MNPs samples. 7 of 20 Nanomaterials 2020, 10, 1499 Table 1. Microstructure of magnetite nanoparticles (MNPs). Table 1. Microstructure of magnetite nanoparticles (MNPs). Sample Fe3O4 Fe3O4/APTES Fe3O4/HA Structure Fe2,94O4 Fe2,88O4 Fe2,93O4 DXRD, nm 1 6.9 ± 2.4 9.6 ± 1.4 10.3 ± 1.3 CV, % 2 34 14.5 12.6 DSEM, nm 3 32.1 ± 4.3 24.18 ± 2.8 34.75 ± 4.3 CV, % 13.5 11.6 12.45 1,3 D XRD and D SEM—average particle size calculated by the Scherrer equation and SEM, respectively, ± standard deviation, nm. 2 CV—coeﬃcient of variation characterizing the polydispersity of the system, %. The lattice parameters determined for all samples formulated in this study are smaller than those reported for magnetite 8.396–8.400 Å (ICDD–PDF 19–629), but larger than those for maghemite 8.33–8.34 Å (ICDD–PDF 39–1346). A plausible explanation of this phenomenon could be the process of partial oxidation of Fe2+ during drying and storage resulting in the nonstoichiometric Fe3−δO4 formation where δ can range from zero (stoichiometric magnetite) to 1/3 (completely oxidized) [59]. mples, respectively (Table 1). 3.1. Microstructure of MNPs 3.1. Microstructure of MNPs For magnetite with an ideal Fe2+ content (assuming the Fe3O4 formula), the mineral phase is known as stoichiometric magnetite (x = 0.50). As magnetite becomes oxidized, the Fe2+/Fe3+ ratio (formula 4) decreases (x < 0.50), with this form denoted as nonstoichiometric or partially oxidized magnetite [59]. The stoichiometry can easily be converted to the following relationship: x = Fe2+ Fe3+ = 1 −3δ 2 + 2δ, (4) (4) Finally, the composition of crystalline component of the samples can be assigned as follows: Fe2,94O4, Fe2,88O4, and Fe2,93O4 for the Fe3O4, Fe3O4/APTES, and Fe3O4/HA samples, respectively (Table S6, Supplementary Materials). A decrease in the magnetite content in the sample with a silica shell Fe3O4/APTES can be associated with the oxidation of magnetite in the synthesis process. The coherent-scattering region size was derived from powder XRD data by Scherrer’s method. According to the formulas of Scherrer, Wolfe-Bragg, and formula for calculating the unit cell parameter, the value of the unit cell parameter a and the average particle size is strongly inﬂuenced by the angle at which the reﬂexes were detected (Q) and the full width at half maximum of XRD reﬂexes (FWHM). The original data is a set of points, for which it is rather diﬃcult to determine Q and FWHM exactly. To calculate these parameters more accurately, the baseline points were described using ﬁve mathematical parameters using to R2 and χ2 as standard statistics parameters for ﬁtting. The full width at half maximum of the reﬂections was used for particle size determination. The reﬂections were ﬁtted with ﬁve models: Gauss function, Lorenz function, Voigt function, Pseudo-Voigt function, and Pearson VII function in Origin 2019 Pro (Figure S1, Supplementary Materials). According to R2 and χ2, data for Fe3O4 and Fe3O4/HA samples were most accurately ﬁtted by the Pseudo-Voigt function (R2 = 0.9098 (9) and χ2 = 11.3893 (5), R2 = 0.9361 (2), and χ2 = 10.2240 (8), Table S7 in Supplementary Materials), Fe3O4/APTES—by the Pearson VII function (R2 = 0.9116 (4) and χ2 = 49.9908 (7), Table S7 in Supplementary Materials). Changes in size of nanoparticles upon coating studied by SEM and XRD correlated. Although the spherical particle shape remained constant for all modiﬁcation routes, a slight particle growth can be observed (Figure 3). For XRD, we used a spherical shape factor (0.94), and as a result, the smaller particle diameters were determined from SEM images. mples, respectively (Table 1). 3.1. Microstructure of MNPs 3.1. Microstructure of MNPs The bare magnetite particles exhibit a median particle diameter of 32.1 and 6.9 nm for SEM and XRD analysis, respectively. Such magnetite particles for the magnetic iron oxide nanoparticles are known to be for superparamagnetic MNPS with a high saturation magnetization and a high speciﬁc surface area [60,61]. Nanomaterials 2020, 10, 1499 Nanomaterials 2020 10 x FO 8 of 20 of 20 8 of 20 of 20 (b) (a) (b) (c) (b) Figure 3. SEM images: (a) Fe3O4, (b) Fe3O4/APTES, and (c) Fe3O4/HA and (d) particle size distribution of samples determined from SEM analysis. Figure 3. SEM images: (a) Fe3O4, (b) Fe3O4/APTES, and (c) Fe3O4/HA and (d) particle size distribution of samples determined from SEM analysis. (a) (b) (a) (b) (c) (b) (c) Figure 3. SEM images: (a) Fe3O4, (b) Fe3O4/APTES, and (c) Fe3O4/HA and (d) particle size distribution of samples determined from SEM analysis. Figure 3. SEM images: (a) Fe3O4, (b) Fe3O4/APTES, and (c) Fe3O4/HA and (d) particle size distribution of samples determined from SEM analysis. 3.2. Characteristics of Surface and Hydrodynamic Size Characteristics of surface charging and hydrodynamic size of MNPs define their biological activity. The interval of pH 6–7 is particularly important for our study as it provides proper conditions for bioassay procedures described in Section 3.3. The particle charge measured as zeta potential is presented in Figure 4 for the Fe3O4, Fe3O4/APTES, and Fe3O4/HA nanoparticles. The charge of native magnetite reverses from positive to negative at pH ∼6.3, which may be considered as isoelectric point (IEP) in accordance with literature values [65]. The reactions of surface Fe–OH sites of magnetite, which can lead to formation of positive (Fe–OH2+) and negative (Fe–O−) surface charges, were observed earlier [5]. In the absence of any steric The diameter of the Fe3O4/APTES particles changed to 9.6 and 24.18 nm compared to bare MNPs, whereas the Fe3O4/HA particles demonstrated a diameter gain to 10.3 and 34.75 nm for SEM and XRD, respectively. The particle growth can be explained by the phase distortion of the original inverse spinel, migration of iron ions to the surface, and subsequent oxidation [62]. Indeed, the XRD patterns conﬁrm preservation of the spinel structure during both modiﬁcation processes: content of magnetite decreases from ~80% to ~75% and ~60% for Fe3O4/HA and Fe3O4/APTES, respectively [63]. mples, respectively (Table 1). 3.1. Microstructure of MNPs 3.1. Microstructure of MNPs According to the coeﬃcient of variation CV and standard deviation value σ of samples for SEM, Fe3O4/APTES has the smaller size distribution (11.6%, 2.8) than Fe3O4 (13.5%, 4.33) and Fe3O4/HA (12.45%, 4.33); all samples are considered to be polydisperse [64]. stabilizing layer, the naked Fe3O4, the uncoated samp zeta potential values were measured between +15 3.2. Characteristics of Surface and Hydrodynamic Size Nanomaterials 2020, 10, x FOR PEER REVIEW 9 of 20 Figure 4. Zeta potential of Fe3O4, Fe3O4/APTES, and Fe3O4/HA nanoparticles as a function of pH (0.01 М KCl). Figure 4. Zeta potential of Fe3O4, Fe3O4/APTES, and Fe3O4/HA nanoparticles as a function of pH (0.01 M KCl). Figure 4. Zeta potential of Fe3O4, Fe3O4/APTES, and Fe3O4/HA nanoparticles as a function of pH (0.01 М KCl). Figure 4. Zeta potential of Fe3O4, Fe3O4/APTES, and Fe3O4/HA nanoparticles as a function of pH (0.01 M KCl). It is known that the presence of amino groups on the surface of Fe3O4 should shift the IEP towards higher pH values [12]. The shift of IEP value of Fe3O4/APTES to pН 7 (Figure 4) was attributed to the presence of protonated amino groups on the SiO2 surface [66]. Additionally, the position of zeta potential curve shows fewer negative charges in the alkaline region on the surface of Fe3O4/APTES, as compared to bare Fe3O4. The reactions of surface Fe3O4–O–Si–NH2 sites with H+ and OH− ions lead to the formation of positive (Fe–O–Si–NH3+) surface charges. The zeta potential of the amino-functionalized MNPs sample via APTES varied drastically between +20 and –10 mV with pH in the given region. However, despite the low surface zeta potential as 0 to –10 mV at pH 7–8, aggregation of MNPs is negligible according to the magnitude of the hydrodynamic diameter varying from 250 to 300 nm (see in Figure 5). Presumably, the ethyl groups of APTES provide steric stabilization, generating an electrosterically stabilized system that confers proportionally larger stability as compared to the pure electrostatic stabilization by the protonated amino groups attached chemically to MNPs’ surface at low pH. It is known that the presence of amino groups on the surface of Fe3O4 should shift the IEP towards higher pH values [12]. The shift of IEP value of Fe3O4/APTES to pH 7 (Figure 4) was attributed to the presence of protonated amino groups on the SiO2 surface [66]. Additionally, the position of zeta potential curve shows fewer negative charges in the alkaline region on the surface of Fe3O4/APTES, as compared to bare Fe3O4. The reactions of surface Fe3O4–O–Si–NH2 sites with H+ and OH− ions lead to the formation of positive (Fe–O–Si–NH3+) surface charges. The zeta potential of the amino-functionalized MNPs sample via APTES varied drastically between +20 and −10 mV with pH in the given region. stabilizing layer, the naked Fe3O4, the uncoated samp zeta potential values were measured between +15 3.2. Characteristics of Surface and Hydrodynamic Size p p p showed worse stability than the MNP samples coated with APTES and humic acids (Figure 4). The Fe3O4/APTES sample showed IEP value at pH 7.1, whereas the Fe3O4/HA sample showed IEP value at pH 3. According to [13], APTES has an IEP at pH 10.05. The variations in data for analogous Characteristics of surface charging and hydrodynamic size of MNPs deﬁne their biological activity. The interval of pH 6–7 is particularly important for our study as it provides proper conditions for bioassay procedures described in Section 3.3. samples in different studies probably indicate the dependence of the zeta potential value on the synthesis approach. The particle charge measured as zeta potential is presented in Figure 4 for the Fe3O4, Fe3O4/APTES, and Fe3O4/HA nanoparticles. The charge of native magnetite reverses from positive to negative at pH ∼6.3, which may be considered as isoelectric point (IEP) in accordance with literature values [65]. The reactions of surface Fe–OH sites of magnetite, which can lead to formation of positive (Fe–OH2+) and negative (Fe–O−) surface charges, were observed earlier [5]. In the absence of any steric stabilizing 9 of 20 Nanomaterials 2020, 10, 1499 layer, the naked Fe3O4, the uncoated sample is only prone to electrostatic stabilization. The zeta potential values were measured between +15 and −30 mV for pH 6–8, and the naked sample showed worse stability than the MNP samples coated with APTES and humic acids (Figure 4). The Fe3O4/APTES sample showed IEP value at pH 7.1, whereas the Fe3O4/HA sample showed IEP value at pH 3. According to [13], APTES has an IEP at pH 10.05. The variations in data for analogous samples in diﬀerent studies probably indicate the dependence of the zeta potential value on the synthesis approach. Nanomaterials 2020, 10, x FOR PEER REVIEW 9 of 20 layer, the naked Fe3O4, the uncoated sample is only prone to electrostatic stabilization. The zeta potential values were measured between +15 and −30 mV for pH 6–8, and the naked sample showed worse stability than the MNP samples coated with APTES and humic acids (Figure 4). The Fe3O4/APTES sample showed IEP value at pH 7.1, whereas the Fe3O4/HA sample showed IEP value at pH 3. According to [13], APTES has an IEP at pH 10.05. The variations in data for analogous samples in diﬀerent studies probably indicate the dependence of the zeta potential value on the synthesis approach. stabilizing layer, the naked Fe3O4, the uncoated samp zeta potential values were measured between +15 3.2. Characteristics of Surface and Hydrodynamic Size However, despite the low surface zeta potential as 0 to −10 mV at pH 7–8, aggregation of MNPs is negligible according to the magnitude of the hydrodynamic diameter varying from 250 to 300 nm (see in Figure 5). Presumably, the ethyl groups of APTES provide steric stabilization, generating an electrosterically stabilized system that confers proportionally larger stability as compared to the pure electrostatic stabilization by the protonated amino groups attached chemically to MNPs’ surface at low pH. The humic acids-coated MNPS are characterized by a negative zeta potential (between −35 and −40 mV) in the bioassay pH interval of 5.8–7.6 (Figure 4). For this pH range, the zeta potential value (between −35 and −40 mV) for humic acids-coated MNPS shows the high degree of repulsion between the charged nanoparticles coating by anionic polyelectrolyte humic acids (pK1 = 4.8 and pK2 = 10, the total acidity of the sample is 5 mmol/g of acidic COOH and OH-groups with pK1 = 4.8 and pK2 = 10, respectively) in the dispersion. Thus, Fe3O4/HA MNPs with high zeta potential values provide the system’s stability in a way of combined steric and electrostatic eﬀects integrated with steric hindrance due to the thick layer of macromolecular humic acids adsorbed on MNPs. A negative charge on the surface of Fe3O4/HA in the pH range of 3–10 indicates the complete coverage of the surface of magnetic nanoparticles with humic acids. 10 of 20 y larger attached Nanomaterials 2020, 10, 1499 g stability as compared to hemically to MNPs surface at low pH. Figure 5. Average hydrodynamic diameter of MNPs nanoparticles as a function of pH (0.01 М KCl). Figure 5. Average hydrodynamic diameter of MNPs nanoparticles as a function of pH (0.01 M KCl). e ica y o N s su ace a o p Figure 5. Average hydrodynamic diameter of MNPs nanoparticles as a function of pH (0.01 М Figure 5. Average hydrodynamic diameter of MNPs nanoparticles as a function of pH (0.01 M Figure 5. Average hydrodynamic diameter of MNPs nanoparticles as a function of pH (0.01 М KCl). Figure 5. Average hydrodynamic diameter of MNPs nanoparticles as a function of pH (0.01 M KCl). The humic acids-coated MNPS are characterized by a negative zeta potential (between −35 and −40 mV) in the bioassay pH interval of 5.8–7.6 (Figure 4). stabilizing layer, the naked Fe3O4, the uncoated samp zeta potential values were measured between +15 3.2. Characteristics of Surface and Hydrodynamic Size For this pH range, the zeta potential value (between −35 and −40 mV) for humic acids-coated MNPS shows the high degree of repulsion between The average hydrodynamic diameter of MNPs depends on the value of pH. In the pH range of bioactivity testing (pH = 6–8), the samples of Fe3O4, Fe3O4/APTES, and Fe3O4/HA have an average hydrodynamic diameter of 100–500 nm (Figure 5). For bare Fe3O4, aggregation of nanoparticles becomes signiﬁcant in this pH interval, due to the absence of electrostatic stabilization near IEP, where the surface charge density of particles is very low. The measured hydrodynamic diameter raises to 500 nm (Figure 5), even at low ionic strength. Modiﬁcation of Fe3O4 by HA led to a signiﬁcant decrease of hydrodynamic diameter to ~120 nm at pH 4–10. 3.3. Eﬀects of MNPs on Bioluminescence of Cellular and Enzyme Assay Systems We examined bioeﬀects of three types of MNPs, i.e., the bare magnetite nanoparticles (Fe3O4), and those modiﬁed by silica (Fe3O4/APTES) and humic acids (Fe3O4/HA). Cellular and enzymatic bioluminescent assay systems (Sections 3.3.1 and 3.3.2, respectively) were used to study the bioeﬀects. Suppression of the bioluminescence is concerned with inhibition of membrane and intracellular processes in the bacterial cells or with inhibition of chemical and biochemical reactions in the enzyme system. The results might be considered as a model of MNP eﬀects on microorganisms and their enzymatic processes in natural ecosystems. Additionally, we studied MNP eﬀects under conditions of an oxidative stress, i.e., in the presence of a model oxidizer, Bq (Section 3.3.2.2). A role of ligand type (APTES or HA) in the catalytic activity of the bacterial enzymes was one of our main focuses. 3.3.1. Eﬀects of MNPs on Bacterial Cells Luminescence intensity of bacterial cells was studied in the presence of MNPs; concentrations of bare Fe3O4, Fe3O4/APTES, and Fe3O4/HA varied as shown in Figure 6. The eﬀects for low MNP concentrations (<2 mg/L) were analyzed. The analysis of higher concentrations of MNPs was limited by their solubility in water and the dispersion stability during the time of the experiments. Additionally, we had to avoid the eﬀect of “optic ﬁlter” which limits application of the luminescence signal registration in high optical density solutions and/or light scattering suspensions [58]. 11 of 20 Nanomaterials 2020, 10, 1499 Figure 6. Relative bacterial bioluminescence intensity, Irel, vs. concentration of MNPs. Figure 6. Relative bacterial bioluminescence intensity, Irel, vs. concentration of MNPs. Figure 6. Relative bacterial bioluminescence intensity, Irel, vs. concentration of MNPs. Figure 6 shows that no signiﬁcant inhibition of the bacterial bioluminescence intensity was observed for the presented samples of MNPs; EC-50 values were not found experimentally. This indicates that MNPs did not result into a high toxic impact on bacterial cells in the concentration range applied. Therefore, the native limitation of MNP content in the water dispersions provides a low inhibitory eﬀect on the bacterial luminescence, in contrast to ﬁne solutions of compounds with higher solubility studied earlier, i.e., metals, their oxides, and organic chemicals [39,67]. The authors reported EC-50 values as 3.5–21 mg/L for soluble rare earth elements, ≤3 mg/L for dopant metals (Ni2+, Fe3+), and no toxicity for metal oxides (EC-50 > 500 mg/L). Figure 6 shows that 20% decrease of the bacterial luminescence intensity was observed for bare Fe3O4 (p < 0.05 at C > 10−2 mg/L, Table S4). This indicates a moderate toxicity of bare Fe3O4 for the bacteria. This eﬀect can be explained by a release of surface Fe2+ to media [68,69] with its following oxidation to Fe3+ in water solutions. The similar oxidative eﬀect of Fe3+ on luminous bacteria was demonstrated earlier in [48,67]. The absence of inhibitory eﬀect of silica-coated MNPs (Fe3O4 /APTES) is evident from Figure 6 (p > 0.05). This result might be related to the speciﬁc characteristics of the silica material used for modiﬁcation of Fe3O4 surface: higher hydrophobicity and low redox activity (due to low surface charge with zeta potential interval of +15 to −10 mV). Probably, the silica cover can neutralize the toxic eﬀect of Fe3O4 surface by preventing it from dissolution of surface iron due to covalent binding. 3.3.1. Eﬀects of MNPs on Bacterial Cells A conﬁrmation of this hypothesis was presented previously in [69]: Swindle et al. showed that organic coatings of the MNP surface protects the particles from the dissolution. Humic acids-coated MNPs, Fe3O4/HA, demonstrated 20% inhibition of the bioluminescence intensity (p < 0.05 at C > 10−3 mg/L), similar to bare Fe3O4, Figure 6. Hence, modiﬁcation of magnetite surface with HA did not protect the bacteria from the toxic eﬀect, in contrast to the silica-modiﬁed magnetite. The peculiarity of its eﬀect is a wide concentration interval of 20% inhibition (ca. 5 × 10−3–1 mg/L). Bioeﬀects of humic substances were observed earlier [47,70]. In [48,49], the inherent inhibitory eﬀect of humic substances on luminous bacteria was found at concentrations higher than 10 mg/L. This value shows that the toxic eﬀect of humic acids-coated MNPs on bacterial cells is not deﬁned by the HA, but by the composite of Fe3O4/HA. A membrane activity of Fe3O4/HA probably can be responsible for its toxic eﬀect on the bacteria: the hydrodynamic diameter of Fe3O4/HA (100 nm) is smaller than the other systems’ diameters (300 nm 12 of 20 Nanomaterials 2020, 10, 1499 for Fe3O4/APTES and 500 nm for the bare Fe3O4) providing a higher penetrating ability for the humic acids-coated nanoparticles. Unlike the other systems, the surface charge of the Fe3O4/HA sample is constantly high and negative (−35 to 40 mV) in the biotesting pH range of 6–7 (Figure 4). However, remarkable variations in zeta potential are not observed likely due to the lower sensitivity of the electrophoretic light scattering method in comparison with the cellular sensitivity. Sensitive Mossbauer spectroscopy showed minor changes in composition of solid Fe3O4-HA due to iron ions release [71,72]. As an outline, a moderate toxicity of bare Fe3O4 was found at concentrations < 2 mg/L; it was supposedly attributed to the oxidative eﬀect of the surface and dissolved Fe3+ in the solutions. High negative surface charge, lower size, and membrane activity, as well as iron ions release, are potential reasons for the moderate eﬀect of Fe3O4/HA. The toxic eﬀect of silica-coated Fe3O4 (Fe3O4/APTES) was absent, thus revealing an inertia of the hydrophobic coating. 3.3.2. Eﬀects of the MNPs on Enzyme Reactions of Luminous Bacteria 3.3.2. Eﬀects of the MNPs on Enzyme Reactions of Luminous Bacteria 3.3.2.1. Eﬀects of the MNPs on the Bioluminescence System of Coupled Reactions 3.3.2.1. Eﬀects of the MNPs on the Bioluminescence System of Coupled Reactions Similar to luminous marine bacteria (Section 3.3.1), the luminescence intensity of the bacterial enzymatic system was studied in the presence of Fe3O4, Fe3O4/HA, and Fe3O4/APTES; low nanoparticle’ concentrations (10−13–2 mg/L) were applied (Figure 7). The eﬀective concentrations EC-20 of Fe3O4/HA and Fe3O4/APTES appeared to be comparable—ca. 1 mg/L, whereas the 20% inhibition of the enzymatic bioluminescence by bare Fe3O4 was found in a wide concentration range: 10−11–2 mg/L. Figure 7. Relative enzyme bioluminescence intensity, Irel, vs. concentration of MNPs. Figure 7. Relative enzyme bioluminescence intensity, Irel, vs. concentration of MNPs. Figure 7. Relative enzyme bioluminescence intensity, Irel, vs. concentration of MNPs. Inhibition of the enzyme bioluminescence system by Fe3O4 was statistically reliable (p < 0.05, Table S2), and the inhibitory eﬀect was not concentration dependent in the used range (p < 0.05; Figure 7). Probably, interactions of the enzymes with the surface of Fe3O4 promote the inhibition of the enzymatic reactions. Additionally, a release of iron ions to water media, discussed before [68,69], can contribute to the speciﬁc inhibition of the bioluminescence intensity. Earlier [46,48], the inhibition of the bioluminescence enzymatic system by Fe3+ was demonstrated. At low concentrations (<10−1 mg/L), no inhibition eﬀects were found in the solutions of modiﬁed MNPs: Fe3O4/APTES and Fe3O4/HA (p > 0.05). Hence, coating the MNP surface with polyelectrolytes APTES and HA prevents from the inhibition of the enzyme reactions, probably, by excluding the enzyme interactions with iron atoms of the MNP surface. 13 of 20 Nanomaterials 2020, 10, 1499 13 of 20 It is evident from the data presented that inhibitory ability of Fe3O4/HA diﬀered in cellular and enzymatic assay systems: humic acids coating protected from the enzyme inhibition, but does not protect from the cell suppression (compare Figures 6 and 7). This comparison likely supports a suggestion on the membrane-active mechanism of toxicity of Fe3O4/HA for bacterial cells. On the other hand, the diﬀerence in results could be additionally explained with the acidity regimes in the assay solutions: the enzymatic assay was conducted in a phosphate buﬀer (pH 6.8), while the bacterial assay was carried out in NaCl solution. The pH regimes might be critical for the toxicity of HA-coated MNPs suspension, and they should be further studied. 3.3.2.2. 3.3.2.1. Eﬀects of the MNPs on the Bioluminescence System of Coupled Reactions Eﬀects of MNPs on the Bioluminescence System of Coupled Reactions under Conditions of Oxidative Stress Since the conditions of water puriﬁcation with MNPs might vary, the biological activity of MNP surface under the oxidative stress is of a special interest. The oxidative stress was chemically modeled in the bioluminescence enzymatic assay. To provide this, the bioluminescence kinetics of the enzyme system was studied in solutions of a model organic oxidizer (1,4-benzoquinone, Bq, 8 × 10−6 M) and MNPs. Concentration of MNPs varied at a low concentration range of 10−13–5 mg/L; the eﬀect of “optic ﬁlter” was excluded. The bioluminescence intensity and the induction period were monitored (see Figure 1 in Section 2.3). The relative bioluminescence intensity, IrelBq, and relative induction period, TrelBq, were calculated at diﬀerent concentrations of MNPs and presented in Figures 8 and 9. Figure 8. Relative bioluminescence induction period in 1,4-benzoquinone solution (8 × 10−6 M), TrelBq, vs. concentration of MNPs. Figure 8. Relative bioluminescence induction period in 1,4-benzoquinone solution (8 × 10−6 M), TrelBq, vs. concentration of MNPs. Figure 8. Relative bioluminescence induction period in 1,4-benzoquinone solution (8 × 10−6 M), TrelBq, vs. concentration of MNPs. Figure 8 demonstrates the TrelBq values of MNPs. Bare Fe3O4 and Fe3O4/APTES did not show the reliable deviations from the control samples (p > 0.05; Table S5; Figure 8). This result reveals an independence of the bioluminescence induction period (T0.5, Figure 1) on the MNP concentrations and demonstrates, by this, that the MNPs do not noticeably change the enzymatic redox activity under the conditions of the oxidative stress. 14 of 20 Nanomaterials 2020, 10, 1499 Figure 9. Relative bioluminescence intensity of enzyme system in 1,4-benzoquinone solution (8 × 10−6 M), IrelBq, vs. concentration of MNPs. Figure 9. Relative bioluminescence intensity of enzyme system in 1,4-benzoquinone solution (8 × 10−6 M), IrelBq, vs. concentration of MNPs. Figure 9. Relative bioluminescence intensity of enzyme system in 1,4-benzoquinone solution (8 × 10−6 M), IrelBq, vs. concentration of MNPs. Only humic acids-coated MNPs, Fe3O4/HA (Figure 8), demonstrated low, but reliable deviations from the control samples (TrelBq < 1) (p < 0.05 at C > 5 × 10−9 mg/L). This eﬀect was constant in a wide range of low concentrations, similar to this presented earlier in Figures 6 and 7. Hence, the results demonstrate that Fe3O4/HA additionally increased the oxidative feature of the Bq solutions. 3.3.2.1. Eﬀects of the MNPs on the Bioluminescence System of Coupled Reactions Figure 9 presents the IrelBq values at diﬀerent concentrations of the MNPs. This parameter (Equation (2)) analyses the bioluminescence intensity, I, of the enzymatic system (Figure 1 in Section 3.2); it depends on the combination of physicochemical mechanisms (redox, polar, and apolar interactions) under conditions of the model oxidative stress. The IrelBq values demonstrated the inhibition of the bioluminescence intensity at higher concentrations of all MNPs (p < 0.05; concentration intervals for statistical conﬁdence are presented in Table S3; Figure 9). All MNPs did not show a reliable activation of the bioluminescence enzyme system (i.e., no mitigation of the oxidative stress) at low concentrations; even Fe3O4/APTES showed the p > 0.05 at 10−7–10−1 mg/L. No statistical diﬀerence was found between the eﬀects of bare Fe3O4 and Fe3O4/APTES under the conditions of the oxidative stress (p > 0.05). The parameters of IrelBq, Figure 9, allowed us to determine the eﬀective concentrations of EC-50 using the experimental data, in contrast to the bioluminescence parameters analyzed before (Irel in the bacterial assay, Irel in the enzymatic assay, and TrelBq in Figure 5, Figure 7, and Figure 8, respectively). So, IrelBq appeared to be most sensitive bioluminescence parameter to monitor the eﬀects of MNPs. The EC-50 were determined as 1, 1, and 5 × 10−2 mg/L for the Fe3O4, Fe3O4/APTES, and Fe3O4/HA, respectively. It is seen that humic acids-coated MNPs, Fe3O4/HA, showed maximal inhibitory activity under the oxidative stress. Additionally, in a wide interval of low concentrations (5 × 10−6–5 × 10−1 mg/L), the Fe3O4/HA demonstrated higher ability to inhibit the bioluminescence intensity (Figure 9), similar to the other kinetic bioluminescence parameter of the enzyme system, TrelBq (Figure 8), discussed before. q Comparison of Figures 8 and 9 reveals that the parameter IrelBq is more sensitive to MNPs than TrelBq. Since the TrelBq is attributed to redox characteristics of solutions only [44,45,82], the sensitivity of the IrelBq parameter might be explained by contribution of hydrophobic interactions to the enzymatic activity under the conditions of oxidative stress. As an outline for Section 3.3.2.2: under the conditions of the model oxidative stress (i.e., in the presence of the model organic oxidizer, Bq), all MNPs, bare and modiﬁed, did not reveal an antioxidant activity. 3.3.2.1. Eﬀects of the MNPs on the Bioluminescence System of Coupled Reactions This eﬀect might be explained with a partial hydrolyzing of HA coating in the Bq solutions and, as a result, higher content of oxidized iron (Fe3+, surface and/or free) in the suspensions of Fe3O4/HA, as compared to bare Fe3O4. Peculiarities of these processes are previously discussed in [71–78], as summarized below: Dissolution of the solid phase and an increase in dissolved Fe2+ and/or Fe3+ as a result of incubation of MNPs with humic substances was demonstrated by Sundman et al. in [71]. Authors revealed that the magnetite incubated with native humic substances becomes more oxidized as compared with a control magnetite [71]. The reason for iron ions release from Fe3O4/HA matrix is the supramolecular nature of HA that means associations via weak hydrophobic (van der Waals, π-π, CH-π) and hydrogen bonds [73]. As a result of diﬀerent treatments (including an oxidative stress), HA can be hydrolyzed, thus leading to the destruction of the protective layer of Fe3O4/HA. Derivatives of HA contribute into the iron ions release from magnetite. Both iron ions, Fe2+ and Fe3+, form strong mixed ligand complexes with HA [72,74] and/or enzymes [75]. Therefore, thermodynamically driven dissolution and subsequent complexation reactions between HA, enzymes, and iron ions can be an important reason for Fe3O4/HA dissolution. The dissolution of Fe3O4/HA was also supported by hydrodynamic particle size analysis via DLS showing an increase in the particle size [76,77]. The studies of the Fe3O4/HA bioeﬀects provides an understanding of a role of humic substances in the biogeochemical cycles of iron (Fe2+/Fe3+). These are naturally important processes, since humic substances are main components of dissolved organic matter and, therefore, play an important role in a complex formation of colloidal magnetite particles, redox activity of iron in these particles, and their eﬀect on microbiota. In nature, humic substances are known to be sorbed onto MNPs, change MNP’ surface charges, aﬀect the aggregation and bioavailability [79], interfere with mineral dissolution/precipitation reactions [80], and drive redox reactions [81]. In contrast to Fe3O4/HA, water solutions of HA did not show any low-concentration inhibitory eﬀects under the conditions of the oxidative stress, but opposite, revealed an antioxidant activity and mitigation of the oxidative eﬀect of Bq (i.e., TrelBq > 1) [49,52,58]. Hence, humic acids-coated MNPs, 15 of 20 Nanomaterials 2020, 10, 1499 Fe3O4/HA, show higher oxidative toxicity than its components Fe3O4 or HA under the conditions of the oxidative stress. 3.3.2.1. Eﬀects of the MNPs on the Bioluminescence System of Coupled Reactions Moreover, humic acids-coated MNPs demonstrated the additional inhibitory eﬀect on the enzyme reactions in a wide concentration range (Figures 8 and 9), which can be explained with HA hydroxylation, a destruction of the protective layer, and a higher content of iron Fe3+ in the samples of Fe3O4/HA, as compared to bare Fe3O4 [78]. 4. Conclusions We studied eﬀects of MNPs on luminous marine bacterium and its enzymatic reactions. Three types of MNPs with diﬀerent surface characteristics were under investigation: bare Fe3O4, as well as Fe3O4 modiﬁed with 3-aminopropyltriethoxysilane (Fe3O4/APTES) and humic acids (Fe3O4/HA). Inhibition eﬀects of the MNPs were studied and compared in a low-concentration range (<2 mg/L). Bacterial bioassay showed a moderate toxicity of bare Fe3O4 and Fe3O4/HA. Based on literature data, we hypothesized that the toxicity of bare Fe3O4 is attributed to the oxidative eﬀect of the surface and free Fe3+ in the suspensions. Toxicity of Fe3O4/HA was supposedly concerned with a high negative surface charge, lower size, and membrane activity of the nanoparticles. The enzymatic bioassay revealed inhibitory ability of bare Fe3O4 only. Additionally, the enzymatic assay was applied to study inhibitory ability of the nanoparticles under conditions of a model oxidative stress (i.e., in the solutions of model oxidizer, 1,4-benzoquinone). We demonstrated that the oxidative stress increases a sensitivity of the enzymatic assay system to MNPs: the bioluminescence intensity determined under the conditions of an oxidative stress, IrelBq, was found to be most sensitive parameter to monitor the inhibitory eﬀects of MNPs. Humic acids-coated MNPs (Fe3O4/HA) showed maximal inhibitory eﬀect on the enzyme reactions, probably due to the partial Nanomaterials 2020, 10, 1499 16 of 20 dissociation of HA coating in the oxidant’ solutions and, as a result, higher content of oxidized iron Fe3+ in the suspensions. Silica surface modiﬁcation made the magnetite nanoparticles (Fe3O4/APTES) inert for both bacteria and their enzymes (Figures 6 and 7). Since silica in free and combined forms is a dominant component of many solid soils and soil solutions/dispersions, our results elucidate the biological function of silica in the biogeochemical cycling of iron. Our results show that the bioluminescence assays, cellular and enzymatic, can serve as promising and highly convenient tools to evaluate and compare the bioavailability of Fe3+ ions in natural dispersions of iron-containing nanoparticles—magnetite, ferrihydrite, so on. The following studies should reveal the quantitative dependences of the bioluminescence response vs. a release of Fe3+ from these MNPs. We have already previously detected the signiﬁcant iron dissolution in the presence of Fe3O4 coated by other ligands [83]. Abbreviations Abbreviations APTES 3-aminopropyltriethoxysilane Bq 1,4-benzoquinone CV Coeﬃcient of variation DLS Dynamic light scattering ELS Electrophoretic light scattering FMN Flavin mononucleotide FWHM Full width at a half maximum HA Humic acids IEP Isoelectric point MNPs Magnetite nanoparticles NADH Nicotinamide adenine dinucleotide disodium salt-reduced SEM Scanning electron microscopy XRD X-ray diﬀraction analysis References 1. Ngomsik, A.-F.; Bee, A.; Draye, M.; Cote, G.; Cabuil, V. Magnetic nano-and microparticles for metal removal and environmental applications: A review. Comptes Rendus Chim. 2005, 8, 963–970. [CrossRef] Abbreviations APTES 3-aminopropyltriethoxysilane Bq 1,4-benzoquinone CV Coeﬃcient of variation DLS Dynamic light scattering ELS Electrophoretic light scattering FMN Flavin mononucleotide FWHM Full width at a half maximum HA Humic acids IEP Isoelectric point MNPs Magnetite nanoparticles NADH Nicotinamide adenine dinucleotide disodium salt-reduced SEM Scanning electron microscopy XRD X-ray diﬀraction analysis References 1. Ngomsik, A.-F.; Bee, A.; Draye, M.; Cote, G.; Cabuil, V. Magnetic nano-and microparticles for metal removal and environmental applications: A review. Comptes Rendus Chim. 2005, 8, 963–970. [CrossRef] APTES 3-aminopropyltriethoxysilane Bq 1,4-benzoquinone CV Coeﬃcient of variation DLS Dynamic light scattering ELS Electrophoretic light scattering FMN Flavin mononucleotide FWHM Full width at a half maximum HA Humic acids IEP Isoelectric point MNPs Magnetite nanoparticles NADH Nicotinamide adenine dinucleotide disodium salt-reduced SEM Scanning electron microscopy XRD X-ray diﬀraction analysis 4. Conclusions Supplementary Materials: The following are available online at http://www.mdpi.com/2079-4991/10/8/1499/s1, Figure S1: ﬁtting experimental data by diﬀerent mathematical models; Table S1: statistic data of ﬁtting by diﬀerent mathematical models for SEM, zeta potential, and hydrodynamic size data; Table S2: statistics of relative enzyme bioluminescence intensity (Figure 7); Table S3: statistics of relative bioluminescence intensity of enzyme system in 1,4-benzoquinone solution (Figure 9); Table S4: statistics of relative bacterial bioluminescence intensity (Figure 6); Table S5: statistics of relative bioluminescence induction period in 1,4-benzoquinone solution (Figure 8); Table S6: microstructure of magnetite nanoparticles (MNPs); Table S7: statistical data of ﬁtting by diﬀerent mathematical models. Author Contributions: Investigation, software, formal analysis, L.S.B.; experimental studies using bioluminescence methods, data processing, interpretation, writing and editing the manuscript, E.S.K.; experimental studies using bioluminescence methods, A.G.K.; resources, investigation of samples microstructure using SEM, writing—review and editing, XRD, G.I.D.; resources, data curation, methodology, E.I. and E.T.; conceptualization, general leadership of the work, data analysis, writing—original draft preparation, writing—review and editing K.A.K. and N.S.K. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Russian Foundation for Basic Research (#19-315-50048, #19-33-90149, and #18-29-19003). Acknowledgments: G.I.D. performed this study in accordance with the state tasks, state registration AAAA-A19- 119032690060-9. Conﬂicts of Interest: The authors declare no conﬂicts of interest. 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https://openalex.org/W2913806412	https://dergipark.org.tr/tr/download/article-file/641815	English	null	Length-Weight Relationships and Condition Factors of 15 Fish Species from KizilirmakYesilirmak Shelf Area, the Southeastern Black Sea	Natural and engineering sciences	2,019	cc-by	3,244	* Corresponding Author: Aysun GÜMÜŞ, e-mail: aysung@omu.edu.tr d 01 October 2018, Accepted 28 November 2018, Available online 31 January 201 Received 01 October 2018, Accepted 28 November 2018, Available online 31 J Abstract Length-weight relationships of 15 fish species from Kizilirmak-Yesilirmak Shelf Area, Southeastern Black Sea were described. A total of 21.246 fish specimens were caught between 2009-2014 using bottom and pelagic trawl nets. Parameters of b estimated for these species varied between 2.82 and 3.49, and r2 varied from 0.82 to 0.99. The lowest and the highest condition factors were estimated for Aphia minuta as 0.50±0.005 and Scophthalmus maximus as 1.68±0.014, respectively. Length-Weight Relationships and Condition Factors of 15 Fish Species from Kizilirmak- Yesilirmak Shelf Area, the Southeastern Black Sea Ondokuz Mayıs University, Faculty of Science and Arts, Department of Biology, Samsun, Turkey NESciences, 2019, 4(1): 21-27 NESciences, 2019, 4(1): 21-27 - RESEARCH ARTICLE - Length-Weight Relationships and Condition Factors of 15 Fish Species from Kizilirmak- Yesilirmak Shelf Area, the Southeastern Black Sea Ayşe Van, Aysun Gümüş*, Serdar Süer Ondokuz Mayıs University, Faculty of Science and Arts, Department of Biology, Samsun, Turkey Ab t t Length-Weight Relationships and Condition Factors of 15 Fish Species from Kizilirmak- Yesilirmak Shelf Area, the Southeastern Black Sea Keywords: Length-weight relationships, demersal fish, pelagic fish, Black Sea. Article history: Introduction The length and weight relationship in fisheries biology is a useful and common tool. This relationship is commonly used to generate the biological data. Primarily, the relationship allows to estimate the mean weight from the given length group by establishing a mathematical relationship between two variables (Beyer, 1991; Froese, 2006). LWR can be used for inter-specific and inter- populational morphometric comparison of fish species. It allows estimation of the condition factor. The condition factor informs about the physiological state of the fish in relation to the general welfare and is associated with biological factors such as age, sex, gonadal development, suitability of the environment and fatness (LeCren, 1951). The estimation of population size of a fish stock requires LWRs knowledge of individuals in the population (Dulčić & Kraljević, 1996). The length Natural and Engineering Sciences 22 and weight relationships can also be used to set yield equations in order to estimate the number of fish landed and to make spatial and temporal comparisons (Beverton & Holt, 1957). and weight relationships can also be used to set yield equations in order to estimate the number of fish landed and to make spatial and temporal comparisons (Beverton & Holt, 1957). The LWR equation is used to explain the isometric or allometric growth pattern of organisms and a number of studies have been carried out on length-weight relationships in Black Sea (Kalayci et al., 2007; Ak et al., 2009; Kasapoglu & Duzgunes, 2013; Yankova et al., 2011; Yesilcicek et al.2015; Yildiz et al., 2018 ). The present study reports the length-weight relationships of nine demersal and six pelagic species from the south-eastern Black Sea sampled in commercial and experimental bottom and pelagic trawl operations. Materials and Methods The samples were monthly collected from Kizilirmak-Yesilirmak Shelf area (KYSA) in the south- eastern Black Sea coast within fishing periods of 2009-2014. The captured fish species were as follows with number of specimens in parantheses: Merlangius merlangus (4727), Mullus barbatus (3470), Scophthalmus maximus (512), Gobius niger (1740), Parablennius tentacularis (423), Trachinus draco (164), Gaidropsarus mediterraneus (164), Aphia minuta (308), Pomatoschistus marmoratus (553), Arnoglossus kessleri (251), Sprattus sprattus (4975), Engraulis encrasicolus (830), Trachurus mediterraneus (1595), Alosa immaculata (825) and Pomatomus saltatrix (709). These species were caught via bottom and pelagic nets along the depth ranges from 0 to100 m. Fish specimens were moved to the laboratory and total length (to the nearest 0.1 cm) and total weight (to 0.01 g the nearest) were measured and the sex is recorded. Length-weight relationships for female, male and pooled specimens of 15 species were estimated using the logarithmic form of Equation (1); where, W is the fish body weight in g, TL is the fish total length in cm, a is the intercept and b is the slope of regression (Froese, 2006). W = a × TLb (1) Type of LWR was described using Student’s-t test. The sex ratio was calculated except Aphia minuta, Pomatoschistus marmoratus and Arnoglossus kessleri. A χ2 test was performed to check any significant difference in sex ratio. The ANCOVA test was used to see whether LWRs between sexes is significantly different or not. Fulton’s condition factor was estimated with the Equation (2) (Froese, 2006); where, W is the fish body weight in g, TL is the fish total length in cm. A t test was used to determine whether there is a significant difference between the mean conditions of sexes. All analyses were performed using SPSS v17.0. K = ( W 𝐿3) × 100 (2) Results In the present study, a total of 21.246 specimens belonging to 15 different fish species and 12 families were analyzed to estimate the LWR parameters. Gobiidae was represented by three species and Clupeidae was represented by two. The sample size (N), sex ratio, total length, total weight, parameters of LWRs a and b, 95% confidence interval for b, coefficient of determination (r2) and growth type for each species and condition factor (K) were presented at Table 1. Natural and Engineering Sciences 23 Table 1. Descriptive statistics for the 15 fish species from KYSA, south-eastern Black Sea. (TL: total length; TW: total weight; N: sample size; M: male; F: female; Ʃ: all individuals; min: minimum; max: maximum; a and b: parameters of LWRs; CI: confidence intervals; r2: coefficient of determination; GT: growth type; A+: positive allometric: A-:negative allometric: I: isometric. a new maximum total length according to Fishbase data. Results b b value different from Bayesian LWR estimates in FishBase, K: Fulton’s condition factor) TL (cm) TW(g) Regression parameters for LWR Species F:M N min max min max a b 95 %CI of b r2 GT K±SE Merlangius merlangus (Linnaeus, 1758)  1.31:1 4727 5.1 23.2 0.20 105.44 0.00512 3.1400 3.126- 3.153 0.98 A+ 0.72±0.001 M 1323 7.2 18.0 2.17 42.92 0.00577 3.0937 3.060- 3.127 0.96 A+ 0.73±0.002 F 1727 6.7 23.2 2.39 105.44 0.00571 3.1016 3.076- 3.127 0.97 A+ 0.75±0.002 Mullus barbatus Linnaeus, 1758  1.80:1 3470 5.2 19.6 1.08 75.00 0.00740 3.1267 3.112- 3.140 0.98 A+ 1.00±0.001 M 639 6.9 15.6 3.00 37.61 0.00845 3.0688 3.010- 3.127 0.94 A+ 0.99±0.003 F 1154 7.5 19.6 3.76 75.00 0.00819 3.0855 3.058- 3.112 0.98 A+ 1.02±0.002 Scophthalmus maximus (Linnaeus, 1758)  1:1 512 7.5 81.3 6.53 102000.00 0.01260 3.0796 3.056- 3.103 0.99 A+ 1.68±0.014 M 133 10.8 57.5 19.90 3200.00 0.01217 3.0873 3.021- 3.153 0.98 A+ 1.65±0.020 F 134 8.4 81.3 9.46 102000.00 0.01531 3.0329 2.958- 3.107 0.98 I 1.75±0.050 Gobius niger Linnaeus, 1758  1.08:1 1740 3.6 12.6 0.35 25.19 0.00572 3.2970 3.269- 3.324 0.97 A+ 1.08±0.003 M 674 4.5 12.5 0.89 22.92 0.00803 3.1376 3.085- 3.191 0.95 A+ 1.12±0.005 F 732 4.7 12.6 0.90 25.19 0.00530 3.3496 3.299- 3.400 0.90 A+ 1.10±0.005 Parablennius tentacularis (Brünnich, 1768)  2.59:1 423 4.8 10.8 0.84 12.85 0.00737 3.1477 3.064- 3.229b 0.93 A+ 0.99±0.005 M 103 5.8 10.8 1.85 12.85 0.01050 2.9766 2.825- 3.127 0.93 I 1.00±0.009 F 267 4.8 9.7 0.84 10.91 0.00648 3.2123 3.086- 3.337 0.91 A+ 0.99±0.006 Trachinus draco Linnaeus, 1758  1.06:1 164 4.4 25.5 0.66 115.75 0.00735 3.0055 2.953- 3.058 0.98 I 0.75±0.06 M 45 9.3 18.2 5.41 44.15 0.00526 3.1259 2.953- 3.298 0.97 I 0.74±0.009 F 48 10.1 14.4 7.06 106.09 0.00642 3.0599 2.850- 3.269 0.95 I 0.77±0.014 Gaidropsarus mediterraneus (Linnaeus, 1758)  1.23:1 164 4.5 23.6 0.25 95.30 0.00293 3.2851 3.232- 3.337b 0.98 A+ 0.61±0.007 M 26 11.2 23.6 8.81 95.30 0.00520 3.0693 2.826- 3.312 0.96 I 0.63±0.014 F 32 10.8 21.9 6.73 87.72 0.00154 3.4238 3.357- 3.690 0.98 A+ 0.66±0.017 Aphia minuta (Risso, 1810)  308 2.9 5.8 0.07 1.10 0.00255 3.4906 3.305- 3.674 0.82 A+ 0.50±0.005 Pomatoschistus marmoratus (Risso, 1810)  553 2.5 6.5 0.11 1.81 0.00566 3.0931 3.018- 3.167 0.93 A+ 0.65±0.004 Arnoglossus kessleri Schmidt, 1915  251 2.9 12.8a 0.03 16.70 0.00948 2.9561 2.814- 3.092 0.87 I 0.92±0.010 Sprattus sprattus (Linnaeus,1758)  3.14:1 4975 4.5 12.8 0.49 12.98 0.00463 3.0984 3.077- 3.119 0.95 A+ 0.54±0.001 Natural and Engineering Sciences 24 M 1114 4.5 12.0 0.49 11.54 0.00472 3.0943 3.055- 3.133 0.96 A+ 0.57±0.002 F 1753 4.6 12.1 0.59 12.98 0.00426 3.1534 3.125- 3.180 0.94 A+ 0.59±0.002 Engraulis encrasicolus (Linnaeus, 1758)  1.28:1 830 5.3 14.2 0.62 18.08 0.00467 3.1026 3.063- 3.142 0.97 A+ 0.60±0.002 M 178 6.7 13.4 2.06 13.95 0.00774 2.9072 2.772- 3.042 0.91 I 0.63±0.004 F 229 7.0 14.0 2.10 18.08 0.00787 2.8975 2.791- 3.003 0.93 I 0.61±0.003 Trachurus mediterraneus (Steindachner, 1868)  1:1.24 1595 5.3 18.8 1.34 49.77 0.00758 3.0311 3.005- 3.056 b 0.97 A+ 0.82±0.002 M 382 7.1 18.8 2.85 46.44 0.00922 2.9524 2.899- 3.005 0.97 I 0.82±0.004 F 308 6.9 18.4 3.11 49.77 0.01034 2.9085 2.851- 2.965 0.97 A- 0.82±0.003 Alosa immaculata Bennett, 1835  1.39:1 825 10.1 29.9 6.40 208.45 0.00384 3.1901 3.150- 3.229b 0.97 A+ 0.66±0.002 M 188 12.7 27.0 11.81 129.45 0.00396 3.1883 3.088- 3.288 0.95 A+ 0.68±0.006 F 262 11.1 29.9 8.89 208.45 0.00336 3.2353 3.166- 3.303 0.97 A+ 0.66±0.004 Pomatomus saltatrix (Linnaeus, 1766)  1.45:1 709 9.1 34.6 6.13 227.91 0.00838 3.0415 3.008- 3.074b 0.98 A+ 0.94±0.004 M 179 10.4 25.4 10.2 162.89 0.00812 3.0513 2.975- 3.127 0.97 I 0.94±0.007 F 261 10.7 29.3 10.41 227.91 0.00884 3.0199 2.971- 3.068 0.98 I 0.94±0.005 All regressions scores were highly significant (P<0.001). Results The sample sizes (N) varied between 26 for Gaidropsarus mediterraneus (female) and 4727 for Merlangius merlangus (all individuals). The exponent b values ranged from 2.82 (in male Gaidropsarus mediterraneus) to 3.49 (in Aphia minuta). Only one species, female Trachurus mediterraneus, showed negative allometric (b<3), 12 cases showed isometric (b=3) and remaining cases showed positive allometric patterns (b>3). Coefficient of determination r2 varied between 0.82 and 0.99. The lowest value of r2 was calculated for Aphia minuta. No significant differences were found between the sex rates of Scophthalmus maximus, Gobius niger, Trachinus draco and Gaidropsarus mediterraneus (P>0.05). There was no statistically significant differences between the sexes with respect to LWRs for Gobius niger, Gaidropsarus mediterraneus, Trachurus mediterraneus and Alosa immaculata (P>0.05). The lowest value of K was recorded for Aphia minuta. The mean K was found to be significantly different within sexes of Merlangius merlangus, Mullus barbatus, Gobius niger, Sprattus sprattus and Alosa immaculata. Discussion The present study provided the first reference on LWRs of Aphia minuta in the Black Sea coast of Turkish waters and for Pomatoschistus marmoratus and Arnoglossus kessleri in mid-southern Black Sea coasts. The b value for all species were between 2.5-3.5 ranges reported by Froese (2006). Parameters a and b of LWRs may indicate variation due to sex, season, year, locality, length range and effected by gonad development and factors such as nutritional condition, temperature and Natural and Engineering Sciences 25 salinity (Froese, 2006). For example, the present study reported that different growth patterns between the male and female for eight species. Females of Scophthalmus maximus showed isometric while males of this species showed positive allometric growth patterns. The condition factor varies according to stomach content, size, season, gonad development and sex (LeCren, 1951; Costa & Araújo, 2003; Froese, 2006). This study showed that sexes have different condition patterns. Table 2 is displayed to discuss the results of present study with the LWRs studies’ from different localities. In our study, we defined a positive allometric LWR in all cases of whiting (M. merlangus). In another study conducted along the south-western Black Sea coasts of Turkey, it is determined as isometric for sexes separately and the positive allometric relationship pattern for overall (Yildiz et al., 2018). However, Yesilcicek et al. (2015) reported a positive allometric relationship type for both sexes in the southern Black Sea. Yildiz et al. (2018) described an isometric relationship for the turbot (S. maximus) female and male individuals but in this study the isometry was only described in females. There is no LWR data identified for sexes for tentacled blenny (P. tentacularis) in FishBase. The positive allometry pattern for the species has been described in the coasts of eastern Aegean Sea (N= 15) and of south-western Black Sea (N=27) (Kara et al., 2016; Yildiz et al., 2018). However, in this study the number of individuals were higher and the relationship is defined as isometric in females and positive allometric in males. While the relationship shows a negative allometry in the Aegean Sea (Filiz & Togulga, 2009), it is estimated as isometric and positive allometric in the Black Sea (Ak et., 2009; Yildiz et al., 2018). It seems that the LWR of the black goby (G. niger) is highly variable. Ak et al. (2009) reported a positive allometric LWR for the greater weever (T. Discussion draco) in the eastern Black Sea. In the present study an isometric pattern is recorded. The b value in shore rockling (G. mediterraneus) is different from Bayesian LWR estimates in FishBase. The b value estimated by Kasapoglu & Duzgunes (2013) for the species is outside the range reported by Froese (2006) (b> 3.5). An isometric length-weight relationship was recorded for the shore rockling in the Sea of Marmara (Bök et al., 2011). In the south-western Black Sea, LWR of the species is generally positive allometric (Yildiz et al., 2018). In this study, a positive allometric LWR was defined for marbled goby (P. marmoratus) from 553 individuals. Yildiz et al. (2018) reported an isometric relationship from 13 individuals. Although, the studies have been conducted in a similar sampling region, LWRs defined for sprat (S. sprattus) have varied. The relationships were reported as positive allometric in this study while as negative allometric by Kalayci et al. (2007). The case is similar for anchovy (E. encrasicolus). While Kalayci et al. (2007) described a negative allometric LWR pattern for the species, in this study it is defined as positive allometric for the sexes, and isometric for the pooled data. Different types of relationship were determined for sexes and for the pooled data of horse mackerel (T. mediterraneus) in this study. In Bulgarian Black Sea waters, the relationship was isometric. Bayesian LWR estimates in FishBase is b = 2.96 (2.93 - 2.99) for the species. The record from the south-western Black Sea is in accordance with the findings of this study for the bluefish (P.saltatrix) (Yildiz et al., 2018). On the other hand, Kalayci et al. (2007) reported a negative allometry for sexes and isometry for the pooled data in the south-middle Black Sea waters. Natural and Engineering Sciences 26 Table 2. Data derived from different studies revealing LWR parameters for 15 fish species in Black Sea. Table 2. Data derived from different studies revealing LWR parameters for 15 fish species in Black Sea. Species N Length range a b Location References M.merlangus 2705 7.6-24.2 0.0046 3.195 Southern Black Sea Yesilcicek et al., 2015 M. barbatus 2693 5.3-19.0 0.0074 3.123 Eastern- central Black Sea Kasapoglu & Duzgunes, 2013 S.maximus 224 6.5-51.7 0.0139 3.054 Western Black Sea Yildiz et al., 2018 G. Discussion niger 112 6.80-15.8 0.0180 2.860 South- Eastern Black Sea Kasapoglu, 2016 P.tentacularis 27 5.5-11.0 0.0061 3.263 Western Black Sea Yildiz et al., 2018 T.draco 338 5.0-35.0 0.0040 3.433 Eastern Black Sea Ak et al., 2009 G.mediterraneus 21 10.8-27.1 0.0012 3.616 Eastern and Central Black Sea Kasapoglu & Duzgunes, 2013 P.marmoratus 13 4.9-7.1 0.0050 3.328 Western Black Sea Yildiz et al., 2018 A.kessleri 60 4.3-9.8 0.0210 2.984 Eastern Black Sea Ak et al., 2009 S. sprattus 5087 5.6-12.6 0.0079 2.867 Middle Black Sea Kalayci, et al., 2007 E. encrasicolus 575 8.0-14.7 0.0174 2.601 Middle Black Sea Kalayci, et al., 2007 T. mediterraneus 1312 9.2-19.0 0.0089 2.900 Eastern Black Sea Sahin, et al., 2009 A. immaculata 36 9.9-28.4 0.0049 3.142 Western Black Sea Yildiz et al., 2018 P. saltatrix 25 12.5-20.2 0.0092 3.005 Eastern and Central Black Sea Kasapoglu & Duzgunes, 2013 † There is no information about LWR for A.minuta in the Black Sea Acknowledgements This study was supported by the Republic of Turkey Ministry of Food, Agriculture, and Livestock (project number HAYSUD/2010/09/01/04) and was performed by TAGEM/SUMAE under the name ‘Monitoring of Trawl Fisheries in The Black Sea’. Ak, O., Kutlu, S., & Aydin, I. (2009). Length-weight relationship for 16 fish species from the Eastern Black Sea, Türkiye. Turkish Journal of Fisheries and Aquatic Sciences, 9(1), 125- 126. References Ak, O., Kutlu, S., & Aydin, I. (2009). Length-weight relationship for 16 fish species from the Eastern Black Sea, Türkiye. Turkish Journal of Fisheries and Aquatic Sciences, 9(1), 125- 126. Ak, O., Kutlu, S., & Aydin, I. (2009). Length-weight relationship for 16 fish species from the Eastern Black Sea, Türkiye. Turkish Journal of Fisheries and Aquatic Sciences, 9(1), 125- 126. Beverton, R.J.H. & Holt, S.J. (1957). On the dynamics of exploited fish populations. Fish Invest. Minist. Agric. Fish Food G.B. (2 Sea Fish). 19, 533 p. Beverton, R.J.H. & Holt, S.J. (1957). On the dynamics of exploited fish populations. Fish Invest. Minist. Agric. Fish Food G.B. (2 Sea Fish). 19, 533 p. Natural and Engineering Sciences 27 Beyer, J.E. (1991). On length-weight relationships: Part II. Computing mean weights from length statistics. Fishbyte, 9: 50-54. Beyer, J.E. (1991). On length-weight relationships: Part II. Computing mean weights from length statistics. Fishbyte, 9: 50-54. Costa, M. R. D., & Araújo, F. G. (2003). Length-weight relationship and condition factor of Micropogonias furnieri (Desmarest)(Perciformes, Sciaenidae) in the Sepetiba Bay, Rio de Janeiro State, Brazil. Revista Brasileira de Zoologia, 20(4), 685-690. Dulčić, J., & Kraljević, M. (1996). Weight-length relationship for 40 fish species in the Eastern Adriatic (Croation waters). Fisheries Research, 28, 243-251. Froese, R. (2006). Cube law, condition factor and weight-length relationship: History, meta- analysis and recommendations. Journal of Applied Ichthyology, 22, 241–253. doi:10.1111/j.1439-0426.2006.00805.x j Kalayci, F., Samsun, N., Bilgin, S., & Samsun, O. (2007). Length-weight relationship of 10 fish species caught by bottom trawl and midwater trawl from the middle Black Sea, Turkey. Turkish Journal of Fisheries and Aquatic Sciences, 7, 33-36. Kasapoglu, N. (2016). Age, growth and mortality rates of discard species (Uranoscopus scaber, Neogobius melanostomus and Gobius niger) in the Black Sea. Ege Journal of Fisheries and Aquatic Sciences, 33(4), 397-403. doi: 10.12714/egejfas.2016.33.4.14 Kasapoglu, N., & Duzgunes, E. (2013). Length-weight relationships of marine species caught by five gears from the Black Sea. Mediterranean Marine Science, 15(1), 95-100. doi: http://dx.doi.org/10.12681/mms.463 Kara, A., Saglam, C., & Acarli, D. (2016). Length-weight relationships of fish captured by wire pots in Izmir Bay (eastern Aegean Sea, Turkey). 2nd International Congress on Applied Ichthyology & Aquatic Environment, 10 - 12 November, Messolonghi, Greece. LeCren, E.D. (1951). The length–weight relationships and seasonal cycle in gonad weight and condition in the perch (Perca fluviatilis). Journal of Animal Ecology, 20, 201–219. 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https://openalex.org/W4322719201	https://link.springer.com/content/pdf/10.1007/s00167-023-07352-9.pdf	English	null	Verasense sensor-assisted total knee arthroplasty showed no difference in range of motion, reoperation rate or functional outcomes when compared to manually balanced total knee arthroplasty: a systematic review	Knee surgery, sports traumatology, arthroscopy	2,023	cc-by	5,239	Abstract Purpose The aim of this systematic review was to investigate the clinical and functional knee outcomes after Verasense sensor-assisted total knee arthroplasty (VA TKA), and to compare these outcomes, where possible, with those from manu- ally balanced total knee arthroplasty (MB TKA). Methods A systematic literature search following PRISMA guidelines was conducted on PubMed, Embase, Medline and Scopus from the beginning of January 2012 until the end of June 2022, to identify potentially relevant articles for this review. Selection was based on the following inclusion criteria: full text English- or German-language clinical studies, published in peer-reviewed journals, which assessed clinical and functional outcomes following VA TKA. Not original research, pre- prints, abstract-only papers, protocols, reviews, expert opinion papers, book chapters, surgical technique papers, and studies pertaining only to unicondylar knee arthroplasty (UKA) or patellofemoral arthroplasty (PFA) were excluded. Several scores (Knee Society Score [KSS], Oxford Knee Score [OKS], Western Ontario and McMaster Universities Osteoarthritis Index [WOMAC], Knee injury and Osteoarthritis Outcome Score—4 subscales [KOOS4] and Physical Function—Computerised Adaptive Testing [PF˗CAT]), alongside postoperative measurements of range of motion [ROM], reoperation rates and the rate of manipulation under anaesthesia [MUA]) were used to evaluate clinical and functional outcomes. The quality of included papers, except randomised control trials (RCTs), was evaluated using the Methodological Index for Non-Randomised Studies (MINORS). For the assessment of included RCTs, the Jadad Scale was used.i Results The literature search identified 243 articles. After removing duplicates, 184 papers were included in the initial screening process. Fourteen of them met all the inclusion criteria following the selection process. Mean MINORS for non- comparative studies value was 11.5 (11–12), and for comparative studies 18.2 (13–21). Mean Jadad Scale score was 3.6 (2–5). Outcomes from a total number of 3633 patients were evaluated (mean age at surgery 68.5 years [32–88 years]). In terms of clinical outcomes, the overwhelming majority of studies observed an improvement after VA TKA, but no statisti- cally significant difference in ROM and reoperation rate when compared to MB TKA. On the other hand, lower rates of MUA have been described in the VA TKA group. Keywords Sensor assisted · Total knee arthroplasty · Total knee replacement · Verasense · Clinical outcomes · Functional outcomes · Intraoperative sensors · Systematic review Knee Surgery, Sports Traumatology, Arthroscopy (2023) 31:1851–1858 https://doi.org/10.1007/s00167-023-07352-9 Knee Surgery, Sports Traumatology, Arthroscopy (2023) 31:1851–1858 https://doi.org/10.1007/s00167-023-07352-9 KNEE KNEE Manuel‑Paul Sava1,2 · Hitomi Hara3 · Leica Alexandra1,2 · Rolf W. Hügli4 · Michael T. Hirschmann1,2 Manuel‑Paul Sava1,2 · Hitomi Hara3 · Leica Alexandra1,2 · Rolf W. Hügli4 · Michael T. Hirschmann1,2 Received: 5 December 2022 / Accepted: 10 February 2023 / Published online: 1 March 2023 © The Author(s) 2023 Extended author information available on the last page of the article Materials and methods A systematic literature search was conducted on PubMed, Embase, Medline and Scopus to identify potentially relevant articles published between June 2012 and June 2022. Terms including “intraoperative sensor”, “sensor technology”, “sensor assisted”, “Verasense”, “pressure sensor”, “force sensor”, “sensor balancing”, “total knee arthroplasty”, “knee revision”, “knee replacement”, “revision arthro- plasty”, “TKA” and “TKR” were searched for in both title and abstract. Detailed information regarding the used search strategy can be found in “Additional file 1”. Abstract An increase in postoperative clinical and functional scores values, when compared to the preoperative ones, has been reported in both groups, although no statistically significant difference between them has been observed.i Conclusion The use of Verasense pressure sensors in TKA leads to no significant improvement in ROM, reoperation rate or functional outcomes, when compared to the standard manually balancing technique. However, lower rates of MUA have been described in the VA TKA group. These findings highlight the importance of tools being able to measure ligament stresses or joint pressure for achieving an optimally balanced knee. Level of evidence III. Keywords Sensor assisted · Total knee arthroplasty · Total knee replacement · Verasense · Clinical outcomes · Functional outcomes · Intraoperative sensors · Systematic review :(0123 1 23456789) 3 Knee Surgery, Sports Traumatology, Arthroscopy (2023) 31:1851–1858 1852 Abbreviations TKA Total knee arthroplasty BMI Body mass index VA Verasense assisted MB Manually balanced UKA Unicondylar knee arthroplasty PFA Patellofemoral arthroplasty PROMs Patient-reported outcome measures KSS Knee Society Score OKS Oxford Knee Score KOOS4 Knee Injury and Osteoarthritis Outcome Score—4 subscales WOMAC Western Ontario and McMaster Universities Arthritis Index PF-CAT Physical Function—Computerised Adaptive Test ROM Range of motion MINORS Methodological Index for Non-Randomised Studies RCT Randomised control trial MUA Manipulation under anaesthesia CR Cruciate retaining PS Posterior stabilised the use of sensors [8, 11, 12], whereas others have failed to demonstrate any clinical benefit [18, 25]. Although an opti- mal balance of the medial and lateral joint gaps in flexion and extension has been considered an important determinant of clinical and functional outcomes, improved outcomes when compared with TKAs that are not balanced, have not yet been proven [9, 17]. One of the commercially available intraoperative sensors is the Verasense sensor [17]. Due to its widespread use, and to the fact that the majority of the studies, which report on clinical and functional outcomes following sensor-assisted TKA, are using Verasense, the present study focuses on its use. The aim of this systematic review was to investigate the clinical and functional knee outcomes after Verasense sensor-assisted TKA (VA TKA) and to compare these outcomes where possible with those from manually balanced TKA (MB TKA). The hypothesis was VA TKA does yield satisfactory postoperative clinical and functional outcomes, but not superior to those after MB TKA. Introduction Achieving optimal clinical and functional outcomes after total knee arthroplasty (TKA) depends on a number of fac- tors such as leg alignment, orientation of the components, as well as adequate soft tissue balancing [2, 9, 21, 24]. Assess- ing soft tissue balance is challenging for the surgeon, as the perceived “feeling for balance” is influenced by several elements such as surgical experience, patients’ body mass index (BMI), gender, soft tissue laxity, the degree of joint contracture, as well as the fitness level of the surgeon [14]. Consequently, this might lead to an unbalanced knee as well as further revisions, which both reduce the patients’ qual- ity of life [11]. Hence, the development of new methods to assess intraoperative ligament laxity and balancing is impor- tant [28]. One of the current debated methods is the use of intraoperative sensor technology, which allows surgeons to dynamically quantify in vivo joint laxity and perform a more objective soft tissue balancing [26]. These sensors enable the surgeon to receive objective intraoperative digital feedback [17]. Intraoperative pressure sensors may play an important role in reducing dissatisfaction related to knee stiffness or instability [11]. Despite their ability to improve soft tissue balance, it is not clear whether the use of intraoperative sensors improve clinical and functional outcomes in TKA. Several studies have shown improved early outcomes with i Inclusion criteria were full text English- or German-lan- guage clinical studies, published in peer-reviewed journals that assessed the clinical and functional outcomes following VA TKA. Only original research articles were considered. Preprints, abstract-only studies, protocols, reviews, expert opinion studies, book chapters, surgical technique studies, and studies pertaining only to unicondylar knee arthroplasty (UKA) or patellofemoral arthroplasty (PFA), were excluded. Only articles reporting clinical and functional knee out- comes after VA TKA were included. After collecting all articles and removing duplicates, two authors screened the studies by title and abstract analysis for inclusion. In a second step, selected articles were checked for their eligibility by full text analysis. In case of uncertainty regarding inclusion a third author was consulted. A further manual screening of the reference lists of included articles was done for additional studies. For this review, only studies reporting clinical and functional outcomes were included. Data extraction ROM was evaluated in five studies (Table 1). They reported no statistically significant differences between the VA and MB groups [5, 7, 19, 25, 29]. Mean follow-up time ranged from 3 to 24 months. From the selected publications, title, author, year of publica- tion, study design, level of evidence, number of knees, fol- low-up time, patient demographics, clinical outcome scores and functional outcomes were extracted. Nine studies examined the rate of MUA as an assess- ment of postoperative arthrofibrosis (Table 2). Geller et al. reported that the rate of MUA in the MB TKA group was higher than in the VA TKA group, and that the period between TKA and MUA was on average 13.5 weeks and 7.5 weeks, respectively [15]. Some of the other studies also showed higher rates of MUA in the MB TKA group, but with no significant differences. The mean follow-up time ranged from 6 to 24 months [1, 3, 5, 7, 16, 18, 19, 25, 29]. Search results and characteristics of included studies The literature search identified 243 scientific articles in the initial screening. A total of 11 met all the inclusion criteria following the selection process shown in Fig. 1 (Additional file 2). Median MINORS for non-comparative studies value was 11.5 (11–12), and for comparative studies 18.2 (13–21). Median Jadad Scale score was 3.6 (2–5). Results from a total number of 3633 patients were evaluated (median age at surgery 68.5 years, range 32–88 years). The reported median body mass index (BMI) was 30.8 (17–45). Several scores (KSS, OKS, WOMAC, KOOS4, PF˗CAT), alongside post- operative ROM, reoperation rates and the rate of manipula- tion under anaesthesia (MUA), were used to evaluate clinical and functional outcomes. The detailed characteristics of the included studies are presented in Table 1 (Additional file 3). Introduction 1 3 3 1853 Knee Surgery, Sports Traumatology, Arthroscopy (2023) 31:1851–1858 Endpoints included various postoperative patient-reported outcome measures (PROMs) such as Knee Society Score (KSS), Oxford Knee Score (OKS), Knee Injury and Osteo- arthritis Outcome Score—4 Subscales (KOOS4), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and Physical Function—Computerised Adap- tive Testing (PF˗CAT), as well as range of motion (ROM), rate of arthrofibrosis and reoperation rate. Quality assessment The methodological quality of the included comparative and non-comparative non-randomised clinical studies was assessed by two raters independently, using the Methodo- logical Index for Non-Randomised Studies (MINORS). The global ideal score is 16 for non-comparative studies, and 24 for comparative studies. For the assessment of included randomised control trials (RCTs), the Jadad Scale was used, with the maximum score being 5. The level of evidence of the included studies was also reported. Statistical analysis of cases, %) p value Geller (2016) [7] VA (292) 3 months 4 (1.40%) 0.004 MB (690) 34 (5.00%) Amundsen (2017) [1] VA (75) 12 months 0 (0.00%) n.s MB (225) 8 (3.50%) Chow (2017) [3] VA (57) 6 months 1 (1.80%) n.s MB (57) 3 (5.30%) Cochetti (2020) [5] VA (50) 24 months 1 (2.10%) nm MB (50) 1 (2.10%) Livermore (2018) [16] AN + MB (103) 12 months 2 (1.94%) n.s AN + VA (74) 1 (1.40%) n.s MacDessi (2021) [18] VA (222) 24 months 9 (3.60%) n.s MB (207) 8 (4.30%) MacDessi (2022) [19] VA (144) 6 months 3 (2.40%) nm MB (141) 7 (4.80%) Song (2019) [25] VA (50) 8.8 ± 2.0 months 0 (0.00%) nm MB (50) 0 (0.00%) Wood (2020) [29] VA (76) 12 months 2 (2.63%) nm MB (76) 2 (2.63%) Table 2 Summary of reported rate of MUA SD standard deviation, VA Verasense sensor assisted, MB manually balanced, ROM range of motion, MUA manipulation under anaesthesia, nm not mentioned, IMG intramedullary guided, AN accelerometer navi- gated Livermore et al. and MacDessi et al. reported the reopera- tion rate at a mean follow-up of 12 months and 24 months, respectively, with no significant differences [16, 18]. Three studies evaluated pre- and postoperative OKS (Table 4). Although the 6-month OKS scores were higher in the VA TKA group (p = 0.01), no significant differences were found between the two groups at 1- and 2-year fol- low-ups [3, 5, 29]. Statistical analysis Continuous variables were described with mean and stand- ard deviation or median and range. Categorical variables were reported with absolute and relative frequencies. A p < 0.05 was considered statistically significant. Table 1 Summary of reported flexion Table 1 Summary of reported flexion SD standard deviation, VA Verasense sensor assisted, MB manually balanced, nm not mentioned Author (year) Groups (no.) Follow-up time (mean ± SD) Preoperative value (mean ± SD) p value Postoperative value (mean ± SD, %) p value Geller (2016) [7] VA (292) 3 months 110° < 0.001 1[12]° n.s MB (690) 104.5° 113.2° Cochetti (2020) [5] VA (50) 24 months 112° ± 11.8° n.s 124° ± 11.5° n.s MB (50) 108° ± 11.8° 123° ± 10.7° MacDessi (2022) [19] VA (144) 6 months nm 105.3° ± 12.9° n.s MB (141) 105.3° ± 15.07° Song (2019) [25] VA (50) 6 months 126.6° ± 16.2° nm 132.6° ± 5.3° nm MB (50) 122.7° ± 14.2° 131.8° ± 7.0° Wood (2020) [29] VA (76) 12 months 109.3° ± 11.5° n.s 118.2° ± 9.1° n.s MB (76) 105.4° ± 13.2° 116.2° ± 11.8° 1854 Knee Surgery, Sports Traumatology, Arthroscopy (2023) 31:1851–1858 SD standard deviation, VA Verasense sensor assisted, MB manually balanced, ROM range of motion, MUA manipulation under anaesthesia, nm not mentioned, IMG intramedullary guided, AN accelerometer navi- gated Author (year) Groups (no.) Follow-up time (mean ± SD) Rate (no. Patient‑reported outcome measures Table 3 Summary of reported KSS scores SD standard deviation, nm not mentioned, Δ mean score change, VA Verasense sensor assisted, MB manually balanced, KSS Knee Society Score, nm not mentioned Author (year) Groups KSS type Follow-up time (mean ± SD) Preoperative score (mean ± SD) p value Postoperative score (mean ± SD) p value Chow (2017) [3] VA (57) KSS total 6 months Δ63 (nm) Δ52 (nm) < 0.001 MB (57) Cochetti (2020) [5] VA (50) KSS total 24 months 64.4 ± 3.9 n.s 165.7 ± 7.3 n.s MB (50) 65 ± 4.0 166.3 ± 3.9 Song (2019) [25] VA (50) KSS total 6 months 79.7 ± 9.2 nm 169.3 ± 5.9 nm MB (50) 78.7 ± 9.4 166.1 ± 6.1 Wood (2020) [29] VA (76) KSS Clinical 12 months 49.7 ± 13.1 n.s 90.0 ± 11.9 n.s MB (76) 49.6 ± 17.4 92.0 ± 10.2 Table 3 Summary of reported KSS scores SD standard deviation, nm not mentioned, Δ mean score change, VA Verasense sensor assisted, MB manually balanced, KSS Knee Society Score, nm not mentioned SD standard deviation, nm not mentioned, Δ mean score change, VA Verasense sensor assisted, MB manually balanced, KSS Knee Society Score, nm not mentioned 1 3 Knee Surgery, Sports Traumatology, Arthroscopy (2023) 31:1851–1858 Knee Surgery, Sports Traumatology, Arthroscopy (2023) 31:1851–1858 1855 Table 4 Summary of reported OKS scores SD standard deviation, nm not mentioned, Δ mean score change, VA Verasense sensor assisted, MB manu- ally balanced, nm not mentioned, OKS Oxford Knee Score Author (year) Groups Follow-up time (mean ± SD) Preop- erative score (mean ± SD) p value Postop- erative score (mean ± SD) p value Chow (2017) [3] VA (57) 6 months Δ17 (nm) Δ13 (nm) 0.025 MB (57) Cochetti (2020) [5] VA (50) 24 months 19.6 ± 2.9 n.s 41.5 ± 4.1 n.s MB (50) 19.0 ± 2.53 41.1 ± 4.0 Wood (2020) [29] VA (76) 12 months 37.9 ± 7.1 n.s 20.5 ± 5.7 n.s MB (76) 38.8 ± 7.6 19.0 ± 7.0 Table 4 Summary of reported OKS scores SD standard deviation, nm not mentioned, Δ mean score change, VA Verasense sensor assisted, MB manu- ally balanced, nm not mentioned, OKS Oxford Knee Score Table 5 Summary of other reported PROMs SD standard deviation, nm not mentioned, Δ mean score change, VA Verasense sensor assisted, MB manually balanced, WOMAC Western Ontario and McMaster Universities Osteoarthritis Index, KOOS4 Knee Injury and Osteoarthritis Outcome Score—4 subscales, PROMIS nm not mentioned, PF-CAT Physical Function—Computerised Adaptive Test Author (year) Groups Type Follow-up time (mean ± SD) Preoperative score (mean ± SD) p value Postoperative score (mean ± SD) p value MacDessi (2022) [19] VA (144) MB (141) WOMAC 24 months 54.5 ± 18.1 nm 14.0 ± 14.4 nm 54.6 ± 15.7 13.0 ± 13.8 KOOS4 38.1 ± 15.0 82.7 ± 15.1 38.0 ± 14.3 82.1 ± 15.5 MacDessi (2021) [18] VA (222) KOOS4 24 months Δ41.5 ± 16.9 n.s MB (207) Δ42.4 ± 19.4 Song (2019) [25] VA (50) WOMAC 12 months 71.7 ± 2.9 nm 17.0 ± 2.9 nm MB (50) 69.3 ± 3.3 17.0 ± 1.8 Livermore (2020) [16] VA (74) MB (103) KOOS4 12 months 51.7 ± 15.3 nm 78.2 ± 16.7 nm 49.8 ± 11.5 76.8 ± 17.1 PF-CAT nm 45.1 ± 8.1 46.6 ± 6.6 Table 5 Summary of other reported PROMs SD standard deviation, nm not mentioned, Δ mean score change, VA Verasense sensor assisted, MB manually balanced, WOMAC Western Ontario and McMaster Universities Osteoarthritis Index, KOOS4 Knee Injury and Osteoarthritis Outcome Score—4 subscales, PROMIS nm not mentioned, PF-CAT Physical Function—Computerised Adaptive Test The most interesting finding of the present review is the fact that when no pressure sensors were used for TKA, the knee was more likely to undergo a manual manipulation [1, 3, 7, 16, 17, 19, 29]. Patient‑reported outcome measures Three studies have reported preoperative and 1- and 2-year postoperative KOOS and WOMAC after VA and MB TKA [16, 19, 25]. They described improved postop- erative scores in both groups, but with no significant dif- ferences between them. Detailed information can be found in Table 5. Four studies have evaluated pre- and postoperative KSS (Table 3). These studies showed improved postoperative KSS, for both groups (VA TKA and MB TKA), but with no significant differences between the analysed groups at 6 and 12 months [3, 5, 25]. Patient‑reported outcome measures However, although there was clear difference for a higher need for MUA in the conventional groups, the findings were not always statistically significant. This might be due to the laxity differences in the included knees. Many studies have just recently highlighted the importance of the envelope of laxity [9, 10]. In line with the ongoing knee phenotype discussion, with regards to alignment parameters, the identification of different knee phenotypes for the individual envelope of laxity has just begun [20, 27]. When having identified different knee lax- ity types, the question arises how to measure these pre-, intra- and postoperatively. Pre- and postoperatively stress radiographs might play a major role. Intraoperative sensor technology, navigational or robotic devices might also be helpful. However, all technologies available are still in the early childhood and needs to mature to make a real clinical impact in TKA. The biggest obstacle besides availability is Discussions The main findings of this study are that both VA TKA and MB TKA showed improved clinical and functional scores, but that there is no statistically significant differ- ence in between VA TKA and MB TKA [5, 7, 19, 25, 29]. Similar findings were shown for PROMs (OKS, KSS, WOMAC, KOOS4 and PF-CAT) [5, 16, 18, 19, 25, 29]. There is only one exception, which has been also reported by Chow et al. who described a statistically significant dif- ference (p < 0.025) in OKS at 6-month follow-up between VA TKA and MB TKA groups [3]. The VA TKA group scored higher (Δ17 vs Δ13). A similar difference for the KSS (Δ63 vs. Δ52) has also been shown by Chow et al. [3]. However, the findings although significant do not rep- resent clinically relevant differences. In addition, in this study, only the mean change between preoperative OKS/ KSS and 6-month postoperative OKS/KSS and not the absolute values were presented. 1 3 1 1856 Knee Surgery, Sports Traumatology, Arthroscopy (2023) 31:1851–1858 Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s00167-023-07352-9. questionable inter- and intra-observer reliability of all such tools, when measuring stress or pressure in the different joint compartments. Acknowledgements The authors would like to extend their thanks to Silvia Reichl, Iris Spörri and Felix Amsler, for offering their invalu- able research knowledge as guidance, when conducting this systematic review. The findings of this review are in line with the findings of Sun et al. in their metanalysis [26]. The number of included studies was lower than in ours. Since then, many more stud- ies have been published. Another difference lies in the avail- ability and the language of the studies included. In the pre- sent review, no articles published in other languages aside English and German were included. In addition, the articles had to possess identification numbers (i.e. PID, DOI), or these had to be accessible (e.g. in online medical libraries [i.e. PubMed, Scopus, Embase, Medline, Google Scholar] and/or peer-reviewed journals). Author contributions Conceptualization: M-PS, HH, AL and MTH; methodology: M-PS and HH; data curation: M-PS, HH, AL and MTH; writing—original draft preparation: M-PS, HH and AL; writ- ing—review and editing: MTH, M-PS, and HH; supervision: MTH and RWH; project administration: M-PS and HH. All the authors have read and agreed to the published version of the manuscript. Funding Open access funding provided by University of Basel. Discussions This research received no external funding. This study presents a number of limitations. The low number of RCTs analysed in our review (i.e. three) com- bined with the inferior quality of some non-randomised clinical studies limits the level of evidence. The low number of enrolled patients in the majority of analysed studies could also play a major role as the ability to identify a statistically significant difference between study groups is heavily linked to the sample size. Although all present completed studies pertaining to this subject have been included, more RCTs and prospective studies are currently in different stages of completion [4, 23]. Their results are yet to be reported; thus, there is a possibility their future findings might not concur with ours. The variability between included studies regarding surgical techniques, type of prosthesis (i.e. cruci- ate retaining [CR] or posterior stabilised [PS]), endpoints, outcomes, follow-up periods and size of cohorts adds to the heterogeneity of the study samples. In addition, the follow- up period for these studies is relatively short, with no study analysing long-term outcomes (clinical or functional). In the end, this systematic review only assessed studies which included a certain type of intraoperative sensors (Verasense). Several other sensors are available on the market (e.g. eLi- bra, Omnibot), to which our findings may not apply [6, 15]. The number of available studies reporting on outcomes following these sensors is low though, and there is none comparing those outcomes to the ones following MB TKA. Therefore, inclusion of these studies in the review, and a subsequent comprehensive analysis of their findings, has not been performed. Data availability Data is available on request in personal repository. Declarations Conflict of interest MTH is a consultant for Medacta™, Symbios™ and Depuy Synthes™. The rest of the authors declare no conflicts of inter- est. Ethical approval Ethical review and approval were waived for this study because unlike primary research, no new personal, sensitive or confidential information has been collected from participants. Only publicly available documents were used for the systematic review. Informed consent Due to the nature of the study, no “Informed Con- sent” was necessary. 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J Arthroplasty 36(3):953–957 17 MacDessi SJ, Wood JA, Diwan AD, Harris IA, Sensor Bal- ance Study Group (2021) Surgeon-defined assessment is a poor predictor of knee balance in total knee arthroplasty: a prospec- tive, multicenter study. Knee Surg Sports Traumatol Arthrosc 29(2):498–506 Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 18. MacDessi SJ, Cohen DA, Wood JA, Diwan AD, Harris IA (2021) Does the use of intraoperative pressure sensors for knee bal- ancing in total knee arthroplasty improve clinical outcomes? A 1 3 Knee Surgery, Sports Traumatology, Arthroscopy (2023) 31:1851–1858 1858 Manuel‑Paul Sava1,2 · Hitomi Hara3 · Leica Alexandra1,2 · Rolf W. Hügli4 · Michael T. Hirschmann1,2 Manuel‑Paul Sava1,2 · Hitomi Hara3 · Leica Alexandra1,2 · Rolf W. Hügli4 · Michael T. Hirschmann1,2 3 Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7‑5‑1 Kusunoki‑cho, Chuo‑ku, Kobe 650‑0017, Japan * Michael T. Hirschmann michael.hirschmann@unibas.ch 1 Department of Orthopaedic Surgery and Traumatology, Kantonsspital Baselland (Bruderholz, Liestal, Laufen), CH‑4101 Bruderholz, Switzerland 4 Institute of Radiology and Nuclear Medicine, Kantonsspital Baselland (Bruderholz, Liestal, Laufen), CH‑4101 Bruderholz, Switzerland 2 Department of Clinical Research, Research Group Michael T. Hirschmann, Regenerative Medicine and Biomechanics, University of Basel, CH‑4001 Basel, Switzerland 1 3 3
https://openalex.org/W3211799178	http://www.scielo.br/pdf/rsp/v3n1/09.pdf	Portuguese	null	Mortalidade "de fato	null	1,969	cc-by	5,290	Recebido para publicação em 4-3-1969. (1) Da Cadeira de Epidemiologia da Faculdade de Higiene e Saúde Pública da USP — São Paulo, Brasil. MORTALIDADE "DE FATO" E "DE DIREITO" PELAS DIVISÕES POLÍTICO-ADMINISTRATIVAS DO MUNICÍPIO DE SÃO PAULO, NO QÜINQÜÊNIO 1959-1963. (ALGUMAS CONSIDERAÇÕES DE INTERESSE EPIDEMIOLÓGICO E ADMINISTRATIVO) (1) Victório BARBOSA BARBOSA, V. — Mortalidade "de fato" e "de direito" pelas divisões político- -administrativas do município de São Paulo, no quinqüênio 1959-1963. (Algumas considerações de interêsse epidemiológico e administrativo). Rev. Saúde públ., S. Paulo, 3(1): 67-77, jun. 1969. Saúde públ., S. Paulo, 3(1): 67 77, jun. 1969. RESUMO — Pela primeira vez entre nós estuda-se a mortalidade de 1-4 anos pelas divisões político-administrativas do município de São Paulo. De- monstra-se que para êsse estudo, os dados oficiais dos óbitos do município de São Paulo, face às diferentes condições sócio-econômica-sanitárias de cada uma dessas áreas, quando comparadas entre si, não são adequados. Propõe-se a sua substituição pelos "de direito" correspondentes, obtidos pessoalmente pelo autor pelo exame dos atestados de óbito da Capital. Apresentam-se alguns aspectos da mortalidade de 1-4 anos "de direito", segundo os distritos e subdis- tritos do município de São Paulo, que se revestem de importância epidemiológica e administrativa para os nossos sanitaristas, quando, êstes últimos, através do uso dêsses dados, entendidos como um indicador de saúde, têm de planejar, programar e executar os seus trabalhos de saúde pública na nossa comunidade. I N T R O D U Ç Ã O GABALDON 2 (1965), desde há muito, vem chamando a atenção para o grande valor do obituário total, ou da mortali- dade geral, do grupo etário de 1-4 anos como um indicador dos problemas sani- tários de países ou áreas em desenvolvi- mento. Para êle, êsse valor decorre do fato de que o coeficiente de mortalidade geral do grupo de 1-4 anos, entre outras coisas, reflete melhor os efeitos da hosti- lidade do meio e da desnutrição, é o que maior redução tem sofrido em seu valor pela aplicação de métodos sanitários ade- quados e, por fim, é o que tem, nas áreas em desenvolvimento, o mesmo significado que o de mortalidade infantil nas áreas desenvolvidas. MORAES 3 (1957), quando propôs sua curva de mortalidade propor- cional como um indicador de saúde útil para comunidades brasileiras, considerou aquêle grupo etário como uma de suas partes constituintes. Técnicos da Organi- zação Mundial de Saúde4 (1957) consi- deraram os óbitos dêste grupo etário, em conjunto com os de menores de 1 ano, como um indicador capaz de expressar a fôrça de mortalidade por doenças trans- missíveis. Ora, sabemos que o município de São Paulo pode ser considerado, sob o ponto de vista sanitário, como uma área em de- senvolvimento, embora, sob muitos aspec- tos já se pode admití-lo num estágio mais avançado, como mostrou RAMOS 6 (1962). Por conseguinte, os óbitos do grupo etário de 1-4 anos adquirem acentuada impor- tância epidemiológica entre nós. Apesar disso, pouca atenção por parte dos nos- sos autores tem sido dada ao seu estudo na Capital. Prova-o o fato de que na literatura local pudemos encontrar ape- nas quatro trabalhos sôbre êle, três dos quais o estudam com exclusividade e, um dêles, como parte integrante de um te- ma mais amplo. Três dêsses trabalhos (PAULA SOUZA & BORGES VIEIRA 5, 1934; ScHMID7, 1962; VERA8, 1963) louvaram- -se nos dados oficiais dos óbitos de 1-4 anos da Capital — dados de fato —; o outro (BARBOSA1, 1967), o mais recente, nesses dados e, também, nos "de direito" correspondentes. magnitude de cada um dos seus proble- mas sanitários — no local certo e na épo- ca oportuna — para, em têrmos de prio- ridades, planejar, executar e avaliar as atividades desenvolvidas na solução dêsses problemas. No caso particular da morta- lidade geral do grupo etário de 1-4 anos. I N T R O D U Ç Ã O tema dêste trabalho, com muito maior ra- zão se aplica esta verdade. Tal ocorre, em parte, devido ao que dissemos sôbre ela no início dêste trabalho e, em parte, graças ao caráter de especificidade que a mesma apresenta pois, sendo restrita a um determinado e bem definido grupo de idade, seus dados são comparáveis, em todos os aspectos que possam nos interes- sar, quando considerados em várias áreas, ou em diferentes pontos de uma mesma área. Por outro lado, no município de São Paulo há uma extrema heterogeneidade de suas divisões político-administrativas, no que tange aos caracteres epidemio- lógicos dos seus problemas de saúde, às estruturas epidemiológicas locais e às ca- racterísticas sócio-econômica-assistenciais e sanitárias de cada uma dessas divisões. Por isso, a classificação da mortalidade geral por tôdas as idades, e mais ainda a do grupo etário de 1-4 anos, têm entre nós, quando comparadas com as de outras áreas em que aquela heterogeneidade geo- gráfica não ocorre, sua importância aumentada na programática dos trabalhos e atividades de saúde pública. Tal se dá, seguramente, nas fases de planejamento, execução e avaliação dêsses trabalhos, quer na organização, estruturação e loca- lização espacial de suas unidades sanitá- rias, quer na quantidade e qualidade dos trabalhos assistenciais e sanitários desen- volvidos por essas unidades sanitárias. Nesse último trabalho, entre nós pio- neiro no concernente à utilização dos "da- dos de direito" para a caracterização da mortalidade do município de São Paulo, pudemos verificar que os "dados de fato" dos óbitos de 1-4 anos, comparados com os "de direito" correspondentes, são ina- dequados. Isto, porque êles não traduzem a verdadeira situação da mortalidade lo- cal no conjunto dessas idades para todo o município, superestimando-a em 21,1%. Nesse estudo preliminar, e não completo, deixamos de abordar a classificação des- ses óbitos pelas divisões político-adminis- trativas da Capital, pelas razoes que então apontamos. Ora, como sabemos, a distribuição da mortalidade geral por tôdas as idades pe- las divisões de uma área geográfica, no sentido do estabelecimento de indicadores do nível de saúde de cada uma delas, tem, na prática de saúde pública, enorme importância epidemiológica-administrativa. I N T R O D U Ç Ã O Isto poderia nos levar a pensar, à primeira vista, que no caso dos distritos e subdis- tritos da Capital, sem exceção, os dados oficiais, por analogia com o ocorrido em todo o município, superestimariam o ver- dadeiro valor do seu obituário, à conta dos eventos de não residentes. Em reali- dade, isto nem sempre acontece. Senão, vejamos! A distribuição dos recursos médico-hos- pitalares de nossa metrópole, em função das necessidades e demandas de seus dis- tritos e subdistritos, não é proporcional, como seria de se desejar. É, na verdade, quanto às facilidades e ao volume de re- cursos médico-hospitalares que proporciona aos seus moradores, bastante irregular e injusta, favorecendo os de certas áreas em detrimento dos de outras. Êstes recursos, em sua maior parte, estão pràticamente concentrados no distrito sede — o de São Paulo —- em uns poucos subdistritos em tôrno de sua área central; isto ocorre, talvez por tradição alicerçada em um pas- sado em que tal se fazia necessário, tal- vez por falta de dados que tivessem per- mitido, através dos tempos e em sinergis- mo com o acelerado crescimento da nos- sa metrópole, uma sua melhor distribuição geográfica. Esta peculiaridade refere-se particularmente aos grandes nosocomios, tanto aos gerais como aos especializados, sobretudo aos que fornecem, em alta esca- la, serviços médicos e cirúrgicos, além de eficientes, gratuitos. Conseqüentemente, resumindo, os dados oficiais dos óbitos de 1-4 anos (dados de fato) dos distritos e subdistritos do mu- nicípio de São Paulo, disponíveis na prá- tica, anàlogamente ao verificado por BAR- BOSA 1 (1967) para todo o município, não reproduzem fielmente em relação a estas áreas o seu obituário real naquelas idades. Isto porque, se com os dêste cotejados (dados de direito), em certos casos o superestimam, em outras ocasiões o subestimam e, por fim, em certas cir- cunstâncias praticamente o igualam. Além disso, êstes dados oficiais, face ao crité- rio legal de sua apuração pelo "local de ocorrência", são, como vimos, inteiramen- te lacunosos quanto ao obituário dos resi- dentes nas divisões administrativas da Ca- pital, tanto que na literatura local que consultamos não há, até o presente, ne- nhum trabalho a respeito. I N T R O D U Ç Ã O Isto porque, conhecendo-os, as autorida- des sanitárias podem, através do método epidemiológico, estabelecer a natureza e a No mesmo município, além disso, os dados oficiais, do mesmo modo com o que ocorre com os dos óbitos por tôdas as idades, se usados na classificação dos óbi- tos de 1-4 anos, de conformidade com as suas divisões político-administrativas — 7 distritos e 39 subdistritos, de 1959 a 1963 — são, mais uma vez, defeituosos ou omissos. Através dêstes dados, à se- melhança do verificado no obituário de todo o município, ficamos conhecendo o total de óbitos de cada uma destas áreas, mas nada ficamos sabendo, por outro la- do, quanto às magnitudes de duas cate- gorias de óbitos, de cuja soma resulta o referido total, ou seja, a dos residentes e a dos não residentes nessas áreas. Isto poderia nos levar a pensar, à primeira vista, que no caso dos distritos e subdis- tritos da Capital, sem exceção, os dados oficiais, por analogia com o ocorrido em todo o município, superestimariam o ver- dadeiro valor do seu obituário, à conta dos eventos de não residentes. Em reali- dade, isto nem sempre acontece. Senão, vejamos! Federação Brasileira, em busca de trata- mento. Muitos destes doentes nelas fale- cem e são registrados, por lei, em seus cartórios; em decorrência, o obituário de cada uma destas áreas, à custa destes óbi- tos, seguramente estará aumentado. Já em relação aos distritos e subdistritos da paulicéia — sobretudo os da periferia — com pouco, ou mesmo sem nenhum re- curso médico-hospitalar, a skuação assu- me característica completamente diferen- te. Com efeito, nestas eventualidades, con- siderando-se comparativamente o número total de óbitos nêles ocorridos e o número real dêsses eventos que lhes deveria caber — o de residentes — esta última cifra pode ser menor ou aproximadamente igual à primeira. ou omissos. Através dêstes dados, à se- melhança do verificado no obituário de todo o município, ficamos conhecendo o total de óbitos de cada uma destas áreas, mas nada ficamos sabendo, por outro la- do, quanto às magnitudes de duas cate- gorias de óbitos, de cuja soma resulta o referido total, ou seja, a dos residentes e a dos não residentes nessas áreas. I N T R O D U Ç Ã O Em vista de tudo que expusemos até agora, vamos apresentar neste trabalho, fundamentalmente através dos "dados de direito", os óbitos de 1-4 anos do muni- cípio de São Paulo, relativos aos anos de 1959 a 1963, de acôrdo com a sua distri- buição pelos distritos e subdistritos que compõem aquela área. Com isso, podere- mos colocar mais uma pedra no mosaico de estudos que vimos fazendo sôbre o obi- tuário "de direito" de 1-4 anos da Capital; É fácil de se compreender, então, que estas divisões administrativas da Capital se constituem, por excelência, em pontos de convergência de milhares de doentes, de todas as direções e sentidos de nossa metrópole, do Estado e de outras áreas da além disso, ao sanarmos uma velha lacuna dos dados de mortalidade da Capital, esta- remos cumprindo a nossa missão dentro da equipe de saúde pública local pois, como sabemos, cabe ao epidemiologista a elucidação dos pontos obscuros existen- tes nos problemas de saúde da comunida- de em que residimos e trabalhamos; final- mente, teremos a oportunidade de, mais uma vez, fornecer aos nossos administra- dores elementos de real valor prático sô- bre aquela mortalidade que, d'ora em diante, poderão servir-lhes de preciosos guias na execução de seus trabalhos, vi- sando a prevenir as doenças e a promo- ver, conservar e recuperar a saúde da população de 1-4 anos residente na área sob sua jurisdição. pia fiel das informações a respeito con- tidas nas tabelas mestras específicas exis- tentes naquele órgão. Na apresentação de nossos dados, do município de São Paulo excluímos Osas- co, em virtude da Lei n.° 5.285. Êste é, aliás, o critério que vem sendo seguido desde 1959 pelo Departamento de Esta- tística do Estado ao coletar, apurar e apresentar os óbitos ocorridos e registra- dos pelo "lugar de ocorrência" do municí- pio em questão que, até 1958, constituía um dos subdistritos do primeiro distrito do município de São Paulo. Na classificação dos óbitos de 1-4 anos "de direito", segundo os distritos e sub- distritos da Capital, tivemos que proceder com cautela; isto foi devido a certas pe- culiaridades locais, que nos dificultaram essa tarefa, tais como: mais de uma rua com o mesmo nome, ruas designadas por número ou por letras maiúsculas do alfa- beto e, por fim, vias públicas que, come- çando em um subdistrito, vão terminar em outro. I N T R O D U Ç Ã O Para obviarmos essas dificuldades, e mantermos a fidedignidade da classifi- cação que fizemos, lançamos mão de vá- rios recursos, dentre os quais os seguin- tes: informações prestadas pelos indicado- res oficiais (guias) das ruas e pelas plantas especializadas e atualizadas do mu- nicípio; indicações dadas por terceiros que conheciam bem esta ou aquela área da Capital; e, finalmente, quando se fazia necessário e possível, elementos obtidos na verificação pessoal "in loco" dos endere- ços anotados nos atestados de óbito que examinamos. M A T E R I A L E M É T O D O S em 5.434, isto é em 66,0% delas, já esta- vam anotadas, ao lado do nome da rua em que residia o falecido, o nome da Vila, Parque, Jardim, Estrada e Km, bairro, subdistrito ou distrito em que aquela rua se encontrava localizada; b) nos ates- tados dos óbitos ocorridos no "domicílio", o nome dos cartórios em que foram re- gistrados os óbitos, face ao dispositivo da Lei n.° 4.887 que manda registrá-los nos cartórios dos subdistritos ou distritos aos quais pertencem a rua em que o mesmo ocorreu, já nos forneciam diretamente sua localização pelas divisões administrativas da Capital. Matarazzo, Guaianazes, Itaquera, Jaraguá, Parelheiros, Perus e São Miguel Paulista. Êsses coeficientes mostraram-se iguais a 6,0 e 6,2, respectivamente, quando cal- culados com os óbitos "de fato", eviden- ciando, dessa maneira, que a mortalidade de 1-4 anos nesses dois grupos geográficos, acima citados, é pràticamente a mesma. Matarazzo, Guaianazes, Itaquera, Jaraguá, Parelheiros, Perus e São Miguel Paulista. Êsses coeficientes mostraram-se iguais a 6,0 e 6,2, respectivamente, quando cal- culados com os óbitos "de fato", eviden- ciando, dessa maneira, que a mortalidade de 1-4 anos nesses dois grupos geográficos, acima citados, é pràticamente a mesma. p Êste fato, encarado de um ponto de vista epidemiológico, significaria que as estruturas epidemiológicas do 1.° Distrito, — áreas centrais da Capital — e dos de- mais Distritos — áreas periféricas do nos- so município — seriam semelhantes e que, portanto, ambas estariam sendo influen- ciadas pelos mesmos fatores sócio-econô- mico-sanitários e assistenciais, condicio- nantes da magnitude do obituário e da mortalidade, de uma e de outra dessas duas áreas geográficas em estudo. Isto. em termos administrativos, nos levaria a admitir, então, que as nossas autoridades sanitárias, quando do planejamento, pro- gramação e execução das medidas reais executivas, visando a diminuição da mor- talidade observada nessas duas áreas, de- veriam considerá-las em igualdade de con- dições quanto: (a) ao estabelecimento das suas prioridades de saúde pública; (b) a programação e execução das suas ativi- dades sanitárias e médico-assistenciais, quanto a natureza dos trabalhos a serem desenvolvidos, no que diz respeito a loca- lização das suas unidades de serviço — ambulatórios, hospitais, unidades sanitá- rias — e, também, no que concerne à dis- tribuição de seu pessoal, material e re- cursos financeiros. Acreditamos ter podido, dessa maneira, localizar convenientemente, segundo as di- visões político-administrativas da Capital, a grande maioria dos óbitos dêsse grupo etário, nessa época. M A T E R I A L E M É T O D O S Reforça esta nossa crença o fato de que, tratando-se de um grande número de observações, igual a 8.233 óbitos, os erros de localização dos óbitos pelas divisões administrativas da Capital, que porventura tenhamos come- tido, devem estar se compensando, de mo- do a não invalidar a sua fidedignidade e portanto, a sua representatividade em re- lação ao que realmente vem ocorrendo em nosso município, quanto a distribuição dêsses óbitos de acôrdo com os seus dis- tritos e subdistritos. M A T E R I A L E M É T O D O S As considerações feitas no capítulo an- terior levaram-nos a incluir neste trabalho, como material primário ou de base, os "dados de direito" dos óbitos ocorridos no município de São Paulo e, como ma- terial para fins de comparação com êstes últimos, os "dados de fato" corresponden- tes. Uns e outros referem-se ao grupo etário de 1-4 anos de nossa população e aos anos de 1959 a 1963. Todos os dados dos óbitos de 1-4 anos apresentados neste trabalho foram obtidos no Departamento de Estatística do Estado. Obtivemos alí também os dados de po- pulação, de duas maneiras: através de estimativas feitas, a nosso pedido, pela sua Secção de Demografia Estática e Di- nâmica e pela consulta a publicações do referido Departamento. Afora isso, duas características que ti- vemos oportunidade de observar nas ano- tações existentes no item "domicílio do falecido" dos atestados de óbito, aí colo- cados por quem os preencheram, também nos foram de grande auxílio e utilidade na feitura dessa classificação. Queremos nos referir às seguintes: a) das 8.233 declarações de óbito que, como vimos em trabalho anterior, correspondem ao total de óbitos de 1-4 anos "de direito" da Capital, durante os anos de 1959 a 1963, Os óbitos de 1-4 anos do município de São Paulo, para os anos de 1959 a 1963, que constituem a essência dêste estudo, foram todos êles, como vimos, obtidos no Departamento de Estatística do Estado: os "de direito" por meio do exame pes- soal e direto das declarações de óbitos respectivas; os "de fato" através da có- em 5.434, isto é em 66,0% delas, já esta- vam anotadas, ao lado do nome da rua em que residia o falecido, o nome da Vila, Parque, Jardim, Estrada e Km, bairro, subdistrito ou distrito em que aquela rua se encontrava localizada; b) nos ates- tados dos óbitos ocorridos no "domicílio", o nome dos cartórios em que foram re- gistrados os óbitos, face ao dispositivo da Lei n.° 4.887 que manda registrá-los nos cartórios dos subdistritos ou distritos aos quais pertencem a rua em que o mesmo ocorreu, já nos forneciam diretamente sua localização pelas divisões administrativas da Capital. R E S U L T A D O S E D I S C U S S Ã O Na Tabela 1 apresentamos os óbitos de 1-4 anos "de fato" e "de direito", bem como os coeficientes de mortalidade ge- ral por mil habitantes correspondentes, classificados pelas divisões político-admi- nistrativas do município de São Paulo, pa- ra o qüinqüênio de 1959-1963. Tendo calculado os coeficientes de mor- talidade de 1-4 anos "de direito", no pe- ríodo de tempo antes considerado, para o total de óbitos do 1.° Distrito e para o total de óbitos obtido pela reunião dêsses eventos dos sete Distritos restantes do mu- nicípio de São Paulo, os quais, como já demonstramos em trabalho anterior, são os que realmente refletem a situação da mortalidade numa dada área geográfica, encontramos. respectivamente, os se- guintes valores: 4,2 e 13.5. Portanto, na Partindo dos dados dessa Tabela, cal- culamos os coeficientes de mortalidade ge- ral para dois grupos de áreas geográfi- cas do nosso município: o primeiro, cons- tituído pelos 39 subdistritos do 1.° Dis- trito — o de São Paulo— e, o segundo, pelos 7 Distritos restantes — Ermelindo O que acabamos de apontar sôbre a mortalidade pelas divisões administrativas do município de São Paulo, por si só, já é suficiente para demonstrar a inadequa- bilidade dos dados oficiais de óbitos — dados de fato — do município de São Paulo, no que tange ao seu uso para pro- pósitos de saúde pública. Não o é, entre- tanto, no sentido de nos fornecer algumas informações de interêsse epidemiológico, ainda ausentes em nosso meio, devido a própria natureza dos dados oficiais de óbitos da Capital atualmente disponíveis, razão porque, em continuação, iremos apresentá-las, evidentemente baseadas na análise dos óbitos "de direito" da Tabela que, como vimos, são os que refletem a real situação da mortalidade de 1-4 anos, do município de São Paulo, segundo as divisões político-administrativas que o compõem nos 5 anos, de 1959 a 1963. verdade, a mortalidade das áreas peri- féricas do nosso município, confrontada com a das áreas centrais, mostra-se 3,2 vêzes maior, fato êste indicativo de que a estrutura epidemiológica da periferia da Capital, mercê do número muito maior de óbitos ocorridos em seus residentes, quan- do cotejado com o verificado nos mora- dores da região central do município, vê- -se substancialmente mais influenciada pe- la ação dos fatôres da mortalidade. R E S U L T A D O S E D I S C U S S Ã O Como conseqüência lógica dêste fato, podemos concluir que no planejamento, programação e execução das atividades assistenciais e sanitárias prioritárias do município de São Paulo, com vistas a obtenção de um nível de saúde, o melhor possível, para a população nêle residente, devem as nossas autoridades sanitárias considerar, em têrmos de prioridade, os sete Distritos do município, que não o de São Paulo, ou seja os da periferia da Ca- pital. Tal atitude, se tomada, òbviamen- te, evitará o desperdício de esforços e de recursos financeiros, devido a sua inade- quada e inoportuna aplicação em áreas centrais que, graças as suas atuais condi- ções de vida, não os estejam realmente necessitando, em detrimento das áreas mais sofridas e desprotegidas — as peri- ferias — justamente as que maior aten- ção estão a requerer por parte dos nossos sanitaristas. A mortalidade dos distritos da Capital, exceto a do primeiro distrito, considerada individualmente para cada um dêles, apre- sentou valores para os seus coeficientes que variaram desde 11,2 (Jaraguá) até 20,1 (Guaianazes). Fazendo-se a ordena- ção desses coeficientes, em ordem decres- cente de sua grandeza, podemos verificar que os distritos de Guaianazes, Perús e Parelheiros, respectivamente, com coefi- cientes de 20,1, 18,8 e 16,6 por mil habi- tantes, colocaram-se nos três primeiros lu- gares, sendo, portanto, aquêles que devem merecer maior atenção, por parte dos nos- sos administradores sanitários, quando, estes últimos, necessitam, com base nes- ses dados de mortalidade, estabelecer as prioridades locais de saúde pública, com vistas ao desenvolvimento dos seus pro- gramas assistenciais e sanitários em nossa comunidade. Seguem-se-lhes, em ordem decrescente de importância, os distritos de Itaquera, Ermelindo Matarazzo, São Mi- guel Paulista e Jaraguá (Fig. 1). Em virtude do exposto, acreditamos ter podido demonstrar, cabalmente, o quão enganosas, para os propósitos da saúde pública, são as nossas estatísticas oficiais de óbitos, dadas pelos óbitos "de fato", quando consideradas no sentido de propor- cionar elementos para a caracterização epidemiológica das divisões político-admi- nistrativas do município de São Paulo e, por conseguinte, para a tomada de me- didas administrativas, assistenciais e sa- nitárias, por quem de direito, visando ao estabelecimento de condições que permi- tem à sua população, considerada em tô- das as partes que a compõem, gozar de um estado de completo bem-estar físico, mental e social. R E S U L T A D O S E D I S C U S S Ã O No que tange a mortalidade dos sub- distritos do distrito de São Paulo, cujo maior coeficiente — o de Santo Amaro — é pouco menor que o do distrito de mais baixa mortalidade, antes apontado (Jara- cleos de mortalidade no grupo etário e período de tempo considerados: um à les- te, um à noroeste e um ao sul, contendo, cada qual dêles, respectivamente, 5, 3 e 2 dessas divisões administrativas da Ca- pital. Com efeito, os dados da Tabela nos proporcionam elementos que nos permitem verificar que, no período em aprêço, dos 8.233 óbitos "de direito" de 1-4 anos de nossa comunidade, 3.816, isto é, 46,4%, ocorreram nesses três núcleos, o que lhes dá um coeficiente igual a 6,6, cerca de 1,8 vezes maior do que o coeficiente de 3,8 obtido pela soma dos óbitos de todos os demais subdistritos do distrito de São Paulo. Estabelecido, dessa maneira, o papel de destaque dos três núcleos de subdistritos antes citados, no que concerne à distri- buição da mortalidade geral de 1-4 anos "de direito" pelas divisões administrati- vas do distrito de São Paulo, é preciso agora, com vistas a um estudo mais deta- lhado deste problema entre nós, procurar as respostas para as seguintes perguntas: (a) qual a importância relativa de cada um dêsses núcleos, quando comparados entre sí? (b) quais os subdistritos per- tencentes a esses núcleos que, na progra- mática dos trabalhos e atividades da saú- de pública local, devem merecer preferên- cia, face a magnitude daquela mortali- dade? guá), fizemos a ordenação dos seus coefi- cientes e dividímo-los em dois grupos assi- nalados pela mediana. Com êstes dados construímos a Figura 2 e, de sua análise, pudemos chegar a várias conclusões quan- to à importância relativa que os nossos administradores sanitários devem dar a cada uma dessas divisões administrativas da Capital, com base nesses dados de mortalidade, por ocasião do estabeleci- mento das prioridades locais de saúde pú- blica. A Figura 2 discrimina, em relação a cada um dos núcleos de subdistritos que estamos estudando, as áreas administrati- vas em que maior mortalidade no grupo etário de 1-4 anos se verificou no período de 1959 a 1963. R E S U L T A D O S E D I S C U S S Ã O Assim, no núcleo do leste essas áreas foram as de Vila Matilde, Vila Maria, Vila Prudente, Penha de França e Tatuapé; no do noroeste, as da Casa Verde, Nossa Senhora do Ó e Piri- tuba; e, por fim, no do sul, as de Santo Amaro e Capela do Socorro. Ressalta, imediatamente, na observação da Figura 2 que, com exceção do Brás, os subdistritos centralmente localizados no município de São Paulo apresentam coe- ficientes de mortalidade menores do que o da mediana (2,7, Parí), ao passo que os subdistritos colocados perifèricamente, ou, então, os contíguos aos da periferia, apresentam valores, para os seus coefi- cientes, acima da mediana. Dentre êstes últimos, por sua vez, destacam-se três nú- Tomando esta observação como ponto de partida podemos, com o auxílio dos dados apresentados na Tabela, calcular os coeficientes de mortalidade geral de 1-4 anos "de direito" de cada um dos citados núcleos. Isto feito, nos é dado verificar que a cada um daqueles três núcleos, na ordem anteriormente apresen- tada, couberam, respectivamente, os se- guintes valores para os coeficientes pro- curados: 5,8, 6,6 e 11,0. Portanto, des- tacou-se o núcleo do sul, eis que o seu coeficiente, quando comparado aos dos dois outros núcleos, antes citados, mos- trou-se maior, respectivamente, cêrca de 1,9 e 1,7 vêzes. BARBOSA, V. — Actual and official morta- lity rate according to the political-admi- nistrative divisions of the county of São Paulo, Brazil, from 1959 to 1963. (A few considerations of epidemiologic and administrative interest). Rev. Saú- de públ., S. Paulo, 3(1) :67-77, jun. 1969. SUMMARY — The author, for the first time among us, studies the death rate of children from one to four years old through the political-administrative divi- sions of the county of São Paulo. After demonstrating that for this study the of- ficial data on deaths in the county, due to the different social, economical and sani- tary conditions of each of these areas when compared to each other, are not adequate, proposes their substitution by corresponding "official" ones which he personally obtained through the exami- nation of the death certificates in the cap- ital. R E S U L T A D O S E D I S C U S S Ã O He presents below some aspects of the "official" death rate of children from one to four years old in the districts and sub-districts of our county, which take on epidemiological and administra- tive importance for our sanitarians, when these, through the use of this data, taken as a health indicator, must plan, schedule and execute their public health services in our municipality. No que tange à segunda pergunta, an- tes formulada, mais uma vez podemos en- contrar a resposta na Tabela. Esta tabe- la, com efeito, nos mostra que, em ordem decrescente de magnitude de mortalidade em estudo, devem merecer a preferência das nossas autoridades sanitárias, na pro- gramática dos seus trabalhos e atividades de saúde pública no 1.° Distrito do nos- so município, os subdistritos de Santo Amaro, Capela do Socorro, Vila Matilde, Casa Verde, Vila Maria, Nossa Senhora do Ó, Pirituba, Vila Prudente, Penha de França e Tatuapé (Fig. 1). Em virtude de tudo que dissemos até agora, em resumo, podemos concluir que a distribuição geográfica da mortalidade de 1-4 anos do município de São Paulo, pelas divisões político-administrativas dês- te último, a se julgar, pelas razões antes apontadas, dos seus dados de óbitos "de direito", e não dos "de fato", é predomi- nantemente centrífuga, pois, como vimos, os maiores valores dos seus coeficientes correspondem às áreas periféricas do nos- so município. Isto quer dizer, em outras palavras, que na programação das medi- das assistenciais e de alcance sanitário, visando ao combate das causas de morte que atingem a população de 1-4 anos da Capital, bem como à promoção, proteção e recuperação da sua saúde, através de medidas de alcance coletivo e de motiva- ção da população, devem merecer abso- luta prioridade, por parte das nossas auto- ridades sanitárias, as áreas periféricas do nosso município. 5. PAULA SOUZA, G. H. & BORGES VIEI- RA, F. — A mortalidade nas crianças de 1-4 anos e suas principais causas em São Paulo. In: CONFERÊNCIA NACIONAL DE PROTEÇÃO À INFÂNCIA. São Paulo, 1933. São Paulo, Imprensa Oficial do Es- tado, 1934. p. 263-289. 6. RAMOS, R. — Indicadores do nível de saúde: sua aplicação no município de São Paulo, 1894-1959. São Paulo, 1962. 136 p. (Tese de doutoramento — Fac. Hig. Saúde Públ. USP). 7. SCHMID, A. W. — Mortalidade e morbi- dade por doenças transmissíveis no muni- cípio de São Paulo no grupo etário de 0-9 anos (1959-1980). Pediat. prát., S. Paulo, 33:133-162, maio, 1962. 8. VERA, R. Del B. de — Observações sôbre a mortalidade no grupo etário de 1-4 anos, no município de São Paulo durante o período de 1950-1961. São Paulo, 1963. (Tese de doutoramento — Fac. Hig. Saú- de Públ. USP). REFERÊNCIAS BIBLIOGRÁFICAS 1. BARBOSA, V. — Estudo comparativo entre os óbitos "de fato e "de direito" no município de São Paulo. São Paulo, 1967. (Tese de Docência-livre — Fac. Hig. Saúde Públ. USP). 2. GABALDON, A. — Indicaciones dadas por las estadisticas vitales. In: —Una política sanitária. Caracas, Ministério de Sanidad y Asistencia Social, 1965. v. 1, p. 194-223. 3. MORAES, N. de A. — Níveis de saúde de coletividades brasileiras. Rev. Serv. Saúde públ., Rio de Janeiro, 9:215-222, dez. 1957. 4. ORGANIZATION MONDIALE DE LA SANTÉ. Groupe d'étude de la mesure de niveaux de santé. — Rapport... Genève, 1957. (Sér. Rapp. techn., n.° 137). 7. SCHMID, A. W. — Mortalidade e morbi- dade por doenças transmissíveis no muni- cípio de São Paulo no grupo etário de 0-9 anos (1959-1980). Pediat. prát., S. Paulo, 33:133-162, maio, 1962. 8. VERA, R. Del B. de — Observações sôbre a mortalidade no grupo etário de 1-4 anos, no município de São Paulo durante o período de 1950-1961. São Paulo, 1963. (Tese de doutoramento — Fac. Hig. Saú- de Públ. USP).
https://openalex.org/W1997845502	https://hal.science/hal-00856210/file/journal.pone.0016724.PDF	English	null	A Novel fry1 Allele Reveals the Existence of a Mutant Phenotype Unrelated to 5′->3′ Exoribonuclease (XRN) Activities in Arabidopsis thaliana Roots	PloS one	2,011	cc-by	11,229	A novel fry1 allele reveals the existence of a mutant phenotype unrelated to 5’->3’ exoribonuclease (XRN) activities in Arabidopsis thaliana roots Judith Hirsch, Julie Misson, Peter A Crisp, Pascale David, Vincent Bayle, Gonzalo M Estavillo, Hélène Javot, Serge Chiarenza, Allison C Mallory, Alexis Maizel, et al. Judith Hirsch, Julie Misson, Peter A Crisp, Pascale David, Vincent Bayle, Gonzalo M Estavillo, Hélène Javot, Serge Chiarenza, Allison C Mallory, Alexis Maizel, et al. To cite this version: Judith Hirsch, Julie Misson, Peter A Crisp, Pascale David, Vincent Bayle, et al.. A novel fry1 allele reveals the existence of a mutant phenotype unrelated to 5’->3’ exoribonuclease (XRN) activities in Arabidopsis thaliana roots. PLoS ONE, 2011, 6 (2), pp.e16724. 10.1371/journal.pone.0016724. hal-00856210 Distributed under a Creative Commons Attribution 4.0 International License Abstract 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. Funding: This work was supported by an ANR-GENOPLANT grant (RIBOROOT-ANR06 GPLA 011) and the CEA agency. Array hybridizations have been partly supported by RNG (Re´seau National des Ge´nopoles, Evry, France). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: elena.marin@cea.fr . These authors contributed equally to this work. . These authors contributed equally to this work. ¤ Current address: UMR BGPI, Campus International de Baillarguet, TA A54/K, Montpellier, France ¤ Current address: UMR BGPI, Campus International de Baillarguet, TA A54/K, Montpellier, France HAL Id: hal-00856210 https://hal.science/hal-00856210v1 Submitted on 5 Oct 2018 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 Judith Hirsch1¤, Julie Misson1., Peter A. Crisp2., Pascale David1., Vincent Bayle1., Gonzalo M. Estavillo2, He´le`ne Javot1, Serge Chiarenza1, Allison C. Mallory3, Alexis Maizel4, Marie Declerck5, Barry J. Pogson2, Herve´ Vaucheret3, Martin Crespi5, Thierry Desnos1, Marie-Christine Thibaud1, Laurent Nussaume1, Elena Marin1* 1 CEA, DSV IBEB, Laboratoire de Biologie du De´veloppement des Plantes, UMR 6191 CNRS, CEA, Aix-Marseille II, Saint-Paul-lez-Durance, France, 2 ARC Centre of Excellence in Plant Energy Biology, Research School of Biology, Australian National University, Canberra, Australian Capital Territory, Australia, 3 Institut Jean-Pierre Bourgin, UMR 1318, INRA, Versailles, France, 4 Department of Stem Cell Biology, University of Heidelberg, Heidelberg, Germany, 5 Institut des Sciences du Ve´ge´tal, CNRS, Gif sur Yvette, France Abstract Background: Mutations in the FRY1/SAL1 Arabidopsis locus are highly pleiotropic, affecting drought tolerance, leaf shape and root growth. FRY1 encodes a nucleotide phosphatase that in vitro has inositol polyphosphate 1-phosphatase and 39,(29),59-bisphosphate nucleotide phosphatase activities. It is not clear which activity mediates each of the diverse biological functions of FRY1 in planta. Principal Findings: A fry1 mutant was identified in a genetic screen for Arabidopsis mutants deregulated in the expression of Pi High affinity Transporter 1;4 (PHT1;4). Histological analysis revealed that, in roots, FRY1 expression was restricted to the stele and meristems. The fry1 mutant displayed an altered root architecture phenotype and an increased drought tolerance. All of the phenotypes analyzed were complemented with the AHL gene encoding a protein that converts 39-polyadenosine 59-phosphate (PAP) into AMP and Pi. PAP is known to inhibit exoribonucleases (XRN) in vitro. Accordingly, an xrn triple mutant with mutations in all three XRNs shared the fry1 drought tolerance and root architecture phenotypes. Interestingly these two traits were also complemented by grafting, revealing that drought tolerance was primarily conferred by the rosette and that the root architecture can be complemented by long-distance regulation derived from leaves. By contrast, PHT1 expression was not altered in xrn mutants or in grafting experiments. Thus, PHT1 up-regulation probably resulted from a local depletion of Pi in the fry1 stele. This hypothesis is supported by the identification of other genes modulated by Pi deficiency in the stele, which are found induced in a fry1 background. Conclusions/Significance: Our results indicate that the 39,(29),59-bisphosphate nucleotide phosphatase activity of FRY1 is involved in long-distance as well as local regulatory activities in roots. The local up-regulation of PHT1 genes transcription in roots likely results from local depletion of Pi and is independent of the XRNs. Citation: Hirsch J, Misson J, Crisp PA, David P, Bayle V, et al. (2011) A Novel fry1 Allele Reveals the Existence of a Mutant Phenotype Unrelated to 59-.39 Exoribonuclease (XRN) Activities in Arabidopsis thaliana Roots. PLoS ONE 6(2): e16724. doi:10.1371/journal.pone.0016724 Editor: Edward Newbigin, University of Melbourne, Australia Received October 18, 2010; Accepted December 22, 2010; Published February 3, 2011 Copyright: 2011 Hirsch et al. This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2011 Hirsch et al. PLoS ONE \| www.plosone.org Citation: Hirsch J, Misson J, Crisp PA, David P, Bayle V, et al. (2011) A Novel fry1 Allele Reveals the Existence of a Mutant Phen Exoribonuclease (XRN) Activities in Arabidopsis thaliana Roots. PLoS ONE 6(2): e16724. doi:10.1371/journal.pone.0016724 February 2011 \| Volume 6 \| Issue 2 \| e16724 A Novel fry1 Allele Reveals the Existence of a Mutant Phenotype Unrelated to 59-.39 Exoribonuclease (XRN) Activities in Arabidopsis thaliana Roots Judith Hirsch1¤, Julie Misson1., Peter A. Crisp2., Pascale David1., Vincent Bayle1., Gonzalo M. Estavillo2, He´le`ne Javot1, Serge Chiarenza1, Allison C. Mallory3, Alexis Maizel4, Marie Declerck5, Barry J. Pogson2, Herve´ Vaucheret3, Martin Crespi5, Thierry Desnos1, Marie-Christine Thibaud1, Laurent Nussaume1, Elena Marin1* Introduction regulator of both ABA-independent and ABA-dependent stress response pathway ([2], Estavillo and Pogson, personal communi- cation) and is involved in leaf venation patterning [3]. Indepen- dent screens also identified fry1 alleles affecting the regulation of photo-morphogenic processes, including hypocotyl elongation and flowering time [4] and lateral root initiation [5]. In the last ten years, a variety of independent genetic screens have identified defects in the enzyme FIERY1/SAL1 (FRY1). The first fry1 mutants were identified in a genetic screen based on the deregulation of an ABA reporter gene [1]. FRY1 was described as a repressor of ABA-mediated stress signal transduction, as the corresponding mutant presented an increased sensitivity to cold, salt and drought stresses [1]. FRY1 seems to act as a negative Such a diversity of phenotypes could be explained by the complexity of FRY1 activity. FRY1 was originally identified as a bifunctional enzyme presenting both an inositol polyphosphate 1- PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16724 1 February 2011 \| Volume 6 \| Issue 2 \| e16724 FRY1 Activity Unlinked to XRNs Figure 1. Phenotype of the fry1-7 mutant. (A-B) Root cross section of a ten day-old PHT1;4:GUS parental line (A) and the PHT1;4:GUS/fry1-7 mutant (B) after GUS staining. Scale bars, 30 mm. (C) Ten day-old plantlets of the PHT1;4:GUS parental line and the PHT1;4:GUS/fry1-7 mutant, note the reduced root system in the mutant. Scale bar, 10 mm. doi:10.1371/journal.pone.0016724.g001 phosphatase activity that hydrolyses inositol 1,4,5-trisphosphate (IP3) in vitro, complementing a salt sensitive yeast strain [6], and a highly specific 39,(29),59-bisphosphate nucleotide phosphatase activity converting PAP (39-polyadenosine 59-phosphate) into AMP and phosphate (Pi) [6,7]. This latter activity was predicted to negatively impact the amount of PAP available in the cell. Indeed, in a separate paper that focuses on chloroplast to nuclear signaling in leaves, several authors from the current study shown that PAP content and not inositol phosphates are regulated by FRY1/SAL1 (Estavillo and Pogson, personal communication). In vitro, PAP suppresses the activity of the yeast 59-.39 exoribonu- cleases Rat1 and Xrn1 [8]. Thus, the accumulation of PAP in loss- of-function fry1 mutants could inhibit the three Rat1 Arabidopsis orthologs XRN2, XRN3 and XRN4, which are all RNA silencing-suppressors [9]. Indeed, some phenotypes of xrn3, xrn2 xrn3 and xrn4 mutants mimic some fry1 traits such as an altered leaf shape, hypocotyl length and reduction of lateral root initiation [4,5,9]. Map-based cloning identifies a new allele of fry1 The mutation was mapped on chromosome 5 between microsatellite markers 5.74 and 5.80, which define an approxi- mately 110 kb interval containing 29 genes. A transcriptomic analysis showed that a transcript corresponding to At5g63980 (FRY1/HOS2/SAL1) was down-regulated in the mutant to 30% of the level detected in the PHT1;4:GUS parental line. Sequencing of the corresponding locus in the mutant line revealed a point mutation (G to A exchange) at nucleotide position 559 in the donor site of the second intron of the FRY1 genomic sequence (Fig. 2A). This mutation altered the splicing of FRY1 transcripts, as confirmed by the cloning and sequencing of four FRY1 splice Results and Discussion Identification of a mutant deregulating PHT1;4::GUS expression and root development Introduction Nevertheless, the contribution of the roots to the reported alx8 and fry1-1 drought tolerance [2] and the role of XRNs in root morphology and drought tolerance have not been analyzed. Using a reporter gene strategy to identify mutations deregulat- ing the expression of the high affinity phosphate transporter PHT1;4 [10], we identified a novel allele of fry1. In addition to root deregulation of the gene reporter, the mutant exhibited strong root architecture defects and a drought resistance phenotype. Through physiological approaches, grafting experiments and mutant analysis, we show that FRY1 plays a role in long distance signaling to roots through its proposed impact on XRN activities in leaves. In contrast, we reveal a new role for FRY1 in the local regulation of phosphate starvation response genes likely linked to a local depletion of Pi in the root stele. Figure 1. Phenotype of the fry1-7 mutant. (A-B) Root cross section of a ten day-old PHT1;4:GUS parental line (A) and the PHT1;4:GUS/fry1-7 mutant (B) after GUS staining. Scale bars, 30 mm. (C) Ten day-old plantlets of the PHT1;4:GUS parental line and the PHT1;4:GUS/fry1-7 mutant, note the reduced root system in the mutant. Scale bar, 10 mm. doi:10.1371/journal.pone.0016724.g001 Identification of a mutant deregulating PHT1;4::GUS expression and root development variants in the mutant (Fig. 2B). All splice variants encoded truncated forms of the FRY1 protein, suggesting that this mutant, referred to as fry1-7, is a loss-of-function allele. Expression of the FRY1 cDNA under the control of the 35S promoter in the fry1-7 line complemented the root phenotype (Fig. S1A), confirming that the mutation in fry1 was responsible for the root defect. Importantly, the expression of the GUS reporter was also com- plemented in PHT1;4:GUS/fry1-7/35S::FRY1 lines (data not shown) and was indistinguishable from the original PHT1;4:GUS line. An allelism test between fry1-7 and the T-DNA insertion allele fry1-6 (Fig. 2A; [9]) further confirmed that FRY1 was the causal gene (data not shown). When driven by the promoter of the high affinity phosphate transporter gene PHT1;4, the GUS reporter gene is induced by phosphate starvation and primarily expressed in the Arabidopsis root. We screened seedlings for the deregulation of this root- expressed reporter gene, in an EMS-derived population of a transgenic line. In our screening conditions (i.e. on phosphate-rich medium) the expression of this reporter marker was not detectable in roots of the parental line [10]. Ten day-old seedlings from each of the 1400 M2 families were stained and roots were screened for seedlings with detectable GUS expression [11]. We identified a recessive mutant (fry1-7, see below) that constitutively expressed the GUS reporter gene in the central cylinder and the pericycle of the root and in primary root meristems (Fig. 1A, B). This mutant also displayed shorter primary and lateral roots (Fig. 1C). Together with the root developmental defects, fry1-7 mutants displayed the aerial growth and developmental defects previously described for other fry1 alleles, including fry1-6 [2,4,9]. Young rosette leaves were crinkly and presented rounded leaf margins and shorter petioles (Fig. S1B), whereas older leaves were serrated. In addition, when transferred to soil, the mutant was more tolerant to drought stress than the wild type control (see below) and displayed a general delay in growth (Fig. S1C) and flowering time (data not shown). PLoS ONE \| www.plosone.org Altered root architecture in fry1 mutants is due to reduced meristem activity in the PR and to an LR initiation defect PHT1;4 expression level in this line by qRT-PCR. In both leaves and roots, we observed an increase in PHT1;4 transcript levels in the fry1-7 single mutant as compared to the Ws control (Fig. 3A). The induction of PHT1;4 was also detected in the fry1-6 allele (Fig. S3), which confirms that the expression of the PHT1;4:GUS transgene in the PHT1;4:GUS/fry1-7 mutant reflects the activation of the endogenous PHT1;4 gene. Thus, in high phosphate conditions, fry1 mutants show a constitutive induction of PHT1;4 in the central cylinder of the root. Alteration of fry1 root architecture has been recently reported [5], but the description of the root phenotype was limited to lateral root initiation. Our analysis indicated that the root system of the fry1-7 mutant is reduced compared to the parental control line both at the primary root (PR) and the lateral root (LR) levels. Seven days post germination (dpg), the fry1-7 mutant primary root was 37% shorter than its parental line, and the fry1-6 primary root was 32% shorter than the Col PR (Fig. 4A). Quantification of PR growth rate during in vitro development in both the fry1-7 and the fry1-6 mutant alleles revealed a statistically significant difference in growth rate when compared with controls (determined by Student’s t test, P,0.01), which likely explains the growth delay observed in the mutant (Fig. 4B). We tested whether the fry1 mutation stimulates the expression of other genes related to PHT1-4. This phosphate transporter belongs to a multigenic family (the PHT1 gene family) that exhibits a tight co-regulation (in particular during Pi deficiency [12]). We found that PHT1;1 and PHT1;2 (revealed by a common pair of primers), PHT1;7 and PHT1;8 transcripts were also induced in fry1-7 (Fig. 3A, B) as compared to the wild type control. In order to assay if genes modulated by Pi starvation distinct from PHT1 family could also be affected by fry1 mutation, we tested two other markers associated with Pi deficiency in the stele: Pho1H1 [13] and the At1G73010 phosphatase (Fig. S2). Both genes were found significantly induced in roots of the fry1 background (Fig. 3C and Fig. S4C, D). Analyses revealed an absence of obvious alterations in Pi content, uptake or transport capacity of the fry1 mutant (data not shown). Nevertheless, the levels of gene induction measured here by qRT-PCR are substantially lower than those observed during phosphate starvation [12,14]. fry1 stimulates the transcription of several genes induced by Pi starvation in the stele To test whether the GUS expression in PHT1;4:GUS/fry1 was due to the upregulation of the endogenous PHT1,4 gene or specific to the T-DNA reporter construct inserted in PHT1;4, we generated a fry1-7 line devoid of any T-DNA insertion by performing a series of back-crosses. We then measured the PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16724 2 FRY1 Activity Unlinked to XRNs Figure 2. Schematic of the mutant fry1 alleles. (A) FRY1 gene structure and position of fry1 mutations. White boxes represent the exons, the horizontal lines represent the introns and the UTRs. In the first exon (e1) the grey box corresponds to the plastid transit peptide (54 amino acids long) predicted in the TAIR database. Positions of the T-DNA insertion in the fry1-3 and fry1-6 mutants alleles are indicated by triangles. The nature of the untagged fry1 alleles is indicated: Lines indicate point mutations, numbers show the position of amino acids, asterisks indicate stop codons. (B) CDS of the FRY1 locus and structure of different splice variants identified in the fry1-7 mutant. In one of the splice variants, the second intron (i2) has been conserved due to the point mutation in fry1-7. The 3rd exon is marked (e3) to clarify the interpretation of the figure. The protein length indicated for each splice variant includes the 54 amino acids of the transit peptide. Asterisks indicate stop codons. doi:10.1371/journal.pone.0016724.g002 Figure 2. Schematic of the mutant fry1 alleles. (A) FRY1 gene structure and position of fry1 mutations. White boxes represent the exons, the horizontal lines represent the introns and the UTRs. In the first exon (e1) the grey box corresponds to the plastid transit peptide (54 amino acids long) predicted in the TAIR database. Positions of the T-DNA insertion in the fry1-3 and fry1-6 mutants alleles are indicated by triangles. The nature of the untagged fry1 alleles is indicated: Lines indicate point mutations, numbers show the position of amino acids, asterisks indicate stop codons. (B) CDS of the FRY1 locus and structure of different splice variants identified in the fry1-7 mutant. In one of the splice variants, the second intron (i2) has been conserved due to the point mutation in fry1-7. The 3rd exon is marked (e3) to clarify the interpretation of the figure. The protein length indicated for each splice variant includes the 54 amino acids of the transit peptide. Asterisks indicate stop codons. doi:10.1371/journal.pone.0016724.g002 Altered root architecture in fry1 mutants is due to reduced meristem activity in the PR and to an LR initiation defect This suggested that the reduction of Pi level is probably limited. In addition, such variation should be restricted to the root stele and masked by the accumulation of vacuolar Pi in external root cell layers such as cortex and epidermis. It is therefore not surprising that such specific Pi discrepancies could not be detected by available techniques and only visualized by the use of sensitive reporter genes or by PCR techniques. We tested whether the fry1 mutation stimulates the expression of other genes related to PHT1-4. This phosphate transporter belongs to a multigenic family (the PHT1 gene family) that exhibits a tight co-regulation (in particular during Pi deficiency [12]). We found that PHT1;1 and PHT1;2 (revealed by a common pair of primers), PHT1;7 and PHT1;8 transcripts were also induced in fry1-7 (Fig. 3A, B) as compared to the wild type control. Reduced root growth can result from a defect in cell elongation and/or from a decrease in meristem activity. Measuring cortical cell length did not reveal any differences between fry1 alleles and wild type controls (Fig. 4C). However, PR cell number in the proximal meristem (PM) at 7dpg [15] was mildly reduced, although statistically significant, in the fry1-6 and fry1-7 mutants when compared to the wild type PM size (Fig. 4D). These results show that the modified PR growth observed in fry1 is due to a defect in maintenance and/or activity of the root apical meristem. Reduced root growth can result from a defect in cell elongation and/or from a decrease in meristem activity. Measuring cortical cell length did not reveal any differences between fry1 alleles and wild type controls (Fig. 4C). However, PR cell number in the proximal meristem (PM) at 7dpg [15] was mildly reduced, although statistically significant, in the fry1-6 and fry1-7 mutants when compared to the wild type PM size (Fig. 4D). These results show that the modified PR growth observed in fry1 is due to a defect in maintenance and/or activity of the root apical meristem. The fry1 mutation also reduced the LR length (Fig. 4E), the LR density (Fig. 4F) and the LR primordia number (Fig. 4G). Thus, it is likely that the altered root architecture of fry1 mutants is not only due to a delay in growth. PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16724 Altered root architecture in fry1 mutants is due to reduced meristem activity in the PR and to an LR initiation defect Interestingly, LR cortical cell length and PM cell number were comparable among fry1-7 and fry1-6 alleles and the corresponding wild type plants when measured at 14 dpg (data not shown), suggesting that an independent factor limits LR initiation or progression. Auxin is a good candidate for such a The fry1 mutation also reduced the LR length (Fig. 4E), the LR density (Fig. 4F) and the LR primordia number (Fig. 4G). Thus, it is likely that the altered root architecture of fry1 mutants is not only due to a delay in growth. Interestingly, LR cortical cell length and PM cell number were comparable among fry1-7 and fry1-6 alleles and the corresponding wild type plants when measured at 14 dpg (data not shown), suggesting that an independent factor limits LR initiation or progression. Auxin is a good candidate for such a PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16724 PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16724 3 FRY1 Activity Unlinked to XRNs Quantitative real time PCR on the PHT1;7 and PHT1;8 loci in fry1-7 and Ws plantlets. (C) Quantitative real time PCR on the At1G73010 and Pho1H1 loci in fry1-7 and Ws roots. Biological triplicates were performed and all samples were analyzed with technical triplicates. White bars correspond to Ws leaves, pale grey bars to fry1-7 leaves, dark grey bars to Ws roots and black bars to fry1-7 roots. Standard deviations are shown. Figure 3. Expression of phosphate induced genes in leaves and roots of the fry1-7 mutant. (A) Quantitative real time PCR of the PHT1;1&PHT1;2 and PHT1;4 transcripts in fry1-7 and Ws plantlets. (B) PLoS ONE \| www plosone org doi:10.1371/journal.pone.0016724.g003 doi:10.1371/journal.pone.0016724.g003 factor as the fry1 mutant has reduced auxin sensitivity at the level of LR initiation [5]. Nevertheless, this auxin response defect could not explain all fry1 root traits as the fry1 PR exhibited auxin sensitivity similar to wild type (data not shown). The 39,(29),59-bisphosphate nucleotide phosphatase activity complements the root mutant phenotype of fry1 as well as the PHT1;4:GUS induction FRY1 is a bifunctional enzyme whereas AHL (Arabidopsis HAL2-like, At5g54390) is a FRY1 paralog encoding a protein with only the 39,(29),59-bisphosphate nucleotide phosphatase activity in vitro [7]. In order to test whether the 39,(29),59-bisphosphate nucleotide phosphatase activity is sufficient to recover wild type root and PHT1;4 induction level, we used the AHL gene harboring only this activity (i.e. not the inositol polyphosphate 1-phosphatase activity). Overexpression of AHL complemented the root pheno- type of the fry1 mutant (Fig. S1D), indicating that the altered root growth of fry1 mutants is likely to be due to the lack of the FRY1 39,(29),59-bisphosphate nucleotide phosphatase activity. In the AHL overexpressor lines, wild type PHT1-4, Pho1H1 and At1g73010 phosphatase transcript levels were re-established (Fig. S3), further confirming the complementation of the fry1 phenotype by AHL activity. As expected, the overexpression of AHL was able to complement the PHT1;4:GUS induction in fry1 (data not shown). These results strongly suggest that the lack of only the 39,(29),59-bisphosphate nucleotide phosphatase activity is respon- sible for all the phenotypes analyzed in the current study. Interestingly, Kim and von Arnim [4] showed that the 35S:AHL construct complements the aerial phenotypes of fry1-6. In vivo analysis of PAP and IP3 levels in Col and fry1 mutants (Estavillo and Pogson, personal communication) confirm our conclusion that only the lack of the 39 (29),59-bisphosphate nucleotide phosphatase activity of FRY1, and the concomitant PAP accumulation, are responsible for all fry1 mutant phenotypes described here. In roots, the FRY1-GFP fusion protein is mainly located in the inner mature tissues and in meristems Grafting experiments reveal two modes of action for FRY1 Five weeks after grafting, we observed that wild type roots remained small like fry1 roots (Fig. 6G). Conversely, the fry1 roots grew like wild type when grafted on a wild type shoot (Fig. 6H). These grafting experiments indicate that the root growth defect of the fry1 mutant is complemented by the wild type shoot. We can hypothesize that a mobile component produced only in leaves is necessary in the root pericyle to exhibit normal root growth. When the aerial part of a graft is unable to synthesize this mobile component (fry1 scion), the roots are less sensitive to auxin and therefore initiate less LR. In roots, the FRY1-GFP fusion protein is mainly located in the inner mature tissues and in meristems The PHT1;4:GUS expression in the internal cell layers of fry1 roots (Fig. 1B) suggests that FRY1 may be expressed in these tissues. To verify this hypothesis, we transformed the PHT1;4:GUS/ fry1-7 mutant line with a GFP-tagged FRY1 genomic construct (pFRY1:FRY1-GFP). This construct is functional because it comple- mented the root development defects of fry1-7 (data not shown). In the mature part of the roots, the FRY1-GFP fluorescence was detected in all cell layers, except the epidermis (Fig. 5A), with strongly enhanced expression in the pericycle and stele regions of the mature part of the PR. The fusion protein was strongly expressed in the PR meristem and the root cap (Fig. 5B). It was also detected in the LR primordia (Fig. 5C), emerged LR (Fig. 5D) and LR meristems (data not shown). Therefore, the overall FRY1 expression pattern largely overlaps with the PHT1;4:GUS expression pattern observed in a fry1-7 mutant background (Fig. 1B). This suggests that the role of FRY1 on PHT1;4 expression is tissue- specific, as the induction appears limited to the regions where FRY1 shows the highest expression level in planta. Figure 3. Expression of phosphate induced genes in leaves and roots of the fry1-7 mutant. (A) Quantitative real time PCR of the PHT1;1&PHT1;2 and PHT1;4 transcripts in fry1-7 and Ws plantlets. (B) PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16724 February 2011 \| Volume 6 \| Issue 2 \| e16724 4 FRY1 Activity Unlinked to XRNs PLoS ONE \| www plosone org 5 February 2011 \| Volume 6 \| February 2011 \| Volume 6 \| Issue 2 \| e16724 PLoS ONE \| www.plosone.org FRY1 Activity Unlinked to XRNs FRY1 Activity Unlinked to XRNs Figure 4. Root architecture of the fry1 mutants. (A) Primary root length at 7 days post germination (dpg). (B) Growth rate of the primary root at 8, 11 and 14 dpg on MS/10 medium. (C) Primary root cortical cell length. (D) Primary root proximal meristem (PM) cell number at 7 dpg. (E) Diagram plotting total lateral root length vs primary root length of the PHT1;4:GUS line (white squares) and PHT1;4:GUS/fry1-7 mutant (grey circles). 18 to 30 plants were measured per genotype, 10 dpg. (F) LR density of fry1 (number of LR per mm PR) at 16 dpg. (G) Number of LR primordia at early stages (I- V) and late stages (VI-VII), 7 dpg. Grafting experiments reveal two modes of action for FRY1 Conversely, the fry1 roots grew like wild type when grafted on a wild type shoot (Fig. 6H). These grafting experiments indicate that the root growth defect of the fry1 mutant is complemented by the wild type shoot. We can hypothesize that a mobile component produced only in leaves is necessary in the root pericyle to exhibit normal root growth. When the aerial part of a graft is unable to synthesize this mobile component (fry1 scion), the roots are less sensitive to auxin and therefore initiate less LR. To help in the interpretation of these contrasting results, we investigated whether grafting could also restore other known characteristics of fry1 mutants. Wilson et al. [2] have shown that fry1 mutants tolerate drought stress up to 50% longer than wild type controls. We used our different graft combinations to test The expression of FRY1 and PHT1;4 in the root stele led us to examine whether the PHT1;4:GUS induction in fry1 could be complemented by a mobile component moving from the shoot. We took advantage of the PHT1;4:GUS reporter in our fry1-7 allele to examine whether FRY1 acts in a tissue-autonomous way. Micrografting experiments were set up with in vitro plantlets (Fig. 6A), using the parental line (PHT1;4:GUS) and the mutant line (PHT1;4:GUS/fry1-7). As expected in high Pi media, we did not observe any GUS expression in roots of the control PHT1;4:GUS//PHT1;4:GUS grafts (Fig. 6B), whereas those of the control PHT1;4:GUS/fry1-7//PHT1;4:GUS/fry1-7 grafts showed strong GUS staining in the central cylinder and the pericycle (Fig. 6C). Grafting a PHT1;4:GUS scion on a PHT1;4:GUS/fry1-7 root stock (Fig. 6D) generated roots with the PHT1;4:GUS/fry1-7 GUS expression pattern, whereas grafting of a PHT1;4:GUS/fry1-7 scion on a PHT1;4:GUS root stock resulted in roots with the GUS pattern of PHT1;4:GUS plants (Fig. 6E). Therefore, a wild type FRY1 in the shoot does not complement the mutant expression pattern of PHT1;4:GUS in the fry1-7 root stock. The complementation of the fry1 root development phenotype and drought resistance by a wild type scion and the non- complementation of the PHT1;4:GUS induction by the wild type scion indicates that FRY1 regulates different aspects of plant physiology by two different mechanisms. Presumably, a mobile component produced by leaves expressing FRY1 is moving to roots and regulating root development but not PHT1 expression. Then, we tested whether a wild type shoot could complement the root growth phenotype of fry1 (Fig. 6F–I). In roots, the FRY1-GFP fusion protein is mainly located in the inner mature tissues and in meristems The wild type and the mutant in A, B, D and G are significantly different (P,0.01) (Student’s t-test). For A–D and F the white bars correspond to the PHT1;4:GUS parental line, the PHT1;4:GUS/fry1-7 mutant appears in pale grey, Col in dark grey and the fry1-6 mutant in black, as detailed in panel B. For all the analyses, at least three independent experiments gave similar results. Standard deviations are shown. doi:10.1371/journal.pone.0016724.g004 Grafting experiments reveal two modes of action for FRY1 whether this tolerance depends on the root system or on the shoot. Fig. 6J shows than when a wild type scion is grafted on a fry1 root it is just as tolerant to drought as when it is grafted on a wild type root (p.0.1). In contrast, wild-type root-stocks did not adversely affect the tolerance of fry1 scions compared to their endogenous roots (p.0.1). By day 12, whatever the grafting combination, most of the wild type scion plants were dead whereas the fry1 scions survided an additional 3 days on average (p.0.1). These experiments demonstrate that the root genotype does not determine the drought tolerance of the aerial part of the plant, indicating that the lack of FRY1 in the leaves is sufficient for drought tolerance. We therefore investigated whether the drought tolerance phenotype of fry1 was due to the lack of FRY1 39 (29),59- bisphosphate nucleotide phosphatase activity. The fry1-6/ 35S::AHL overexpression line displayed a wild type level of drought tolerance (Fig. 7A), indicating that the drought tolerance of fry1 is due to the lack of 39 (29),59-bisphosphate nucleotide phosphatase activity. FRY1 The expression of FRY1 and PHT1;4 in the root stele led us to examine whether the PHT1;4:GUS induction in fry1 could be complemented by a mobile component moving from the shoot. We took advantage of the PHT1;4:GUS reporter in our fry1-7 allele to examine whether FRY1 acts in a tissue-autonomous way. Micrografting experiments were set up with in vitro plantlets (Fig. 6A), using the parental line (PHT1;4:GUS) and the mutant line (PHT1;4:GUS/fry1-7). As expected in high Pi media, we did not observe any GUS expression in roots of the control PHT1;4:GUS//PHT1;4:GUS grafts (Fig. 6B), whereas those of the control PHT1;4:GUS/fry1-7//PHT1;4:GUS/fry1-7 grafts showed strong GUS staining in the central cylinder and the pericycle (Fig. 6C). Grafting a PHT1;4:GUS scion on a PHT1;4:GUS/fry1-7 root stock (Fig. 6D) generated roots with the PHT1;4:GUS/fry1-7 GUS expression pattern, whereas grafting of a PHT1;4:GUS/fry1-7 scion on a PHT1;4:GUS root stock resulted in roots with the GUS pattern of PHT1;4:GUS plants (Fig. 6E). Therefore, a wild type FRY1 in the shoot does not complement the mutant expression pattern of PHT1;4:GUS in the fry1-7 root stock. Then, we tested whether a wild type shoot could complement the root growth phenotype of fry1 (Fig. 6F–I). Five weeks after grafting, we observed that wild type roots remained small like fry1 roots (Fig. 6G). The xrn2 xrn3 xrn4 triple mutant displays the fry1 lateral root and drought tolerance phenotypes but does not affect primary root It has been proposed that XRN activity is inhibited in a fry1 background [9], likely because of the accumulation of the XRNs inhibitor PAP (Estavillo and Pogson, personal communication). Accordingly, both fry1 and the xrn mutants accumulate RNA intermediates of miRNA-directed post-transcriptional regulation and share common traits [9]. To further analyze the role of XRN in the fry1 phenotype, we generated an xrn2 xrn3 xrn4 triple mutant that was fertile, unlike the sterile xrn2 xrn3 double mutant. Thus To help in the interpretation of these contrasting results, we investigated whether grafting could also restore other known characteristics of fry1 mutants. Wilson et al. [2] have shown that fry1 mutants tolerate drought stress up to 50% longer than wild type controls. We used our different graft combinations to test Figure 5. Pattern of expression of the FRY1-GFP fusion protein in roots. Roots of a fry1-7 mutant complemented with a pFRY1:FRY1:GFP construct were observed by confocal microscopy. (A) Mature root. (B) PR meristem. (C) LR primordium. (D) Emerged LR. Scale bars are 75 mm in A, B and C, and 150 mm in D. doi:10.1371/journal.pone.0016724.g005 Figure 5. Pattern of expression of the FRY1-GFP fusion protein in roots. Roots of a fry1-7 mutant complemented with a pFRY1:FRY1:GFP construct were observed by confocal microscopy. (A) Mature root. (B) PR meristem. (C) LR primordium. (D) Emerged LR. Scale bars are 75 mm in A, B and C, and 150 mm in D. doi:10.1371/journal.pone.0016724.g005 February 2011 \| Volume 6 \| Issue 2 \| e16724 PLoS ONE \| www.plosone.org 6 FRY1 Activity Unlinked to XRNs Figure 6. FRY1 in shoot complements the root growth defects of fry1 but not the expression of the PHT1;4:GUS reporter gene. (A) The grafting junction. Arrow indicates the silicon ring. (B–E) The different graft combinations (scion/root) between the PHT1;4:GUS line and the PHT1;4:GUS line are indicated. Below are the corresponding pictures of a grafted root after the overnight GUS staining. (F–I) Shoot and root phenotypes of the different graft combinations (scion/root) between the wild type and the fry1 mutant, after 4 weeks of growth in soil. Note that the root growth of fry1 is complemented by the wild type shoot (H), but wild type roots display a fry1 phenotype when grafted with a fry1 scion (G). (J) Survival rate after withholding watering of plants with different grafting combinations show that the drought tolerance phenotype of fry1 is determined by the scion genotype. The xrn2 xrn3 xrn4 triple mutant displays the fry1 lateral root and drought tolerance phenotypes but does not affect primary root Data are from one representative experiment out of three. Error bars represent standard error. doi:10.1371/journal.pone.0016724.g006 Figure 6. FRY1 in shoot complements the root growth defects of fry1 but not the expression of the PHT1;4:GUS reporter gene. (A) The grafting junction. Arrow indicates the silicon ring. (B–E) The different graft combinations (scion/root) between the PHT1;4:GUS line and the PHT1;4:GUS line are indicated. Below are the corresponding pictures of a grafted root after the overnight GUS staining. (F–I) Shoot and root phenotypes of the different graft combinations (scion/root) between the wild type and the fry1 mutant, after 4 weeks of growth in soil. Note that the root growth of fry1 is complemented by the wild type shoot (H), but wild type roots display a fry1 phenotype when grafted with a fry1 scion (G). (J) Survival rate after withholding watering of plants with different grafting combinations show that the drought tolerance phenotype of fry1 is determined by the scion genotype. Data are from one representative experiment out of three. Error bars represent standard error. doi:10.1371/journal.pone.0016724.g006 shape, drought tolerance), they do not mimic the induction of the PHT1;4 locus. In addition, a qRT-PCR analysis of the xrn2 xrn3 xrn4 triple mutant confirmed that the XRN activities are not responsible for the up-regulation of PHT1 genes (Fig. S4). Indeed, the assayed mutants (xrn4-6 and the xrn2 xrn3 xrn4) showed the same level of PHT1;4, PHT1;7, Pho1H1 and AT1g73019 transcripts as the Col control. Thus, this analysis further confirmed that the xrn mutations do not mimic the induction of the PHT1;4 locus, nor the general induction of phosphate-starvation genes observed in the fry1 background. the triple xrn2 xrn3 xrn4 mutant facilitated in vitro root analysis without antibiotic selection, which has negative consequences on root development. Although the mechanism for the partial phenotypic rescue is unclear, it suggests that xrn4 mutations act to partially suppress the xrn2 xrn3 phenotypic effects. We found that the lateral root phenotype of the xrn2 xrn3 xrn4 triple mutant was similar to that of fry1 (Fig. 7B), whereas the primary root of the triple mutant was not significantly reduced compared to wild type (Fig. 7C). We also found that the xrn2 xrn3 xrn4 triple mutant tolerated a drought stress like the fry1 mutants (Fig. 7A). Altogether, these results suggest that the pleiotropic phenotype of the fry1 mutants results, at least in part, from a general perturbation in XRN activities. The xrn2 xrn3 xrn4 triple mutant displays the fry1 lateral root and drought tolerance phenotypes but does not affect primary root The inability of xrn mutants to induce PHT1;4 transcription argues in favor of a model whereby FRY1 has two physiological roles for the 39,(29),59-bisphosphate nucleotide phosphatase activity (Modeled in Fig. 8). On one hand, the PAP accumulation in the mutant represses XRN activity, altering various phenotypes linked to the deregulation of the silencing machinery (root architecture, drought tolerance, leaf shape, hypocotyl sensitivity to red light, hormonal sensing and signaling). Indeed, fry1 late flowering, short petioles and hypocotyl hypersensitivity to red light phenotypes are largely mimicked by the xrn2 xrn3 double mutant [4]. The root architecture of the xrn4 single mutant has been described as being similar to that of fry1 [5]. However, only the xrn2 xrn3 xrn4 triple mutant presents fry1-like lateral root architecture defects in our conditions (Figs. 7B, C). The xrn4 mutant presents wild type LR development (Fig. S5A) and a PR PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16724 The PHT1;4:GUS induction in fry1 is unrelated to its action on XRNs We investigated whether the xrn mutations could mimic the induction of PHT1;4:GUS observed in fry1. For this, we crossed the PHT1;4:GUS parental line to the different single, double and triple xrn mutant lines. We confirmed the crosses by checking that the GUS marker was active in L of the F2 when plants were grown in phosphate deficient media (Table 1). Interestingly, in plantlets grown in complete media, we never observed GUS-stained roots (Table 1) demonstrating that although the xrn mutations can mimic many of the fry1 mutant phenotypes (root architecture, leaf February 2011 \| Volume 6 \| Issue 2 \| e16724 February 2011 \| Volume 6 \| Issue 2 \| e16724 7 FRY1 Activity Unlinked to XRNs Figure 7. The xrn2 xrn3 xrn4 triple mutant mimics fry1 drought tolerance and root architecture phenotypes. (A) Dehydration experiment on 4 week-old soil grown plants of the indicated genotypes. Two independent experiments, with 6 to 17 plants per genotype per experiment, gave the same results. (B) Root architecture phenotype of the xrn2 xrn3 xrn4 triple mutant compared to the wild type Col and the fry1-6 mutant at 11 dpg. Scale bar is 20 mm. (C) Primary root length of the same plantlets, at the same age. Note that the length of Col and xrn2 xrn3 xrn4 primary roots are not significantly different. doi:10.1371/journal.pone.0016724.g007 Figure 7. The xrn2 xrn3 xrn4 triple mutant mimics fry1 drought tolerance and root architecture phenotypes. (A) Dehydration experiment on 4 week-old soil grown plants of the indicated genotypes. Two independent experiments, with 6 to 17 plants per genotype per experiment, gave the same results. (B) Root architecture phenotype of the xrn2 xrn3 xrn4 triple mutant compared to the wild type Col and the fry1-6 mutant at 11 dpg. Scale bar is 20 mm. (C) Primary root length of the same plantlets, at the same age. Note that the length of Col and xrn2 xrn3 xrn4 primary roots are not significantly different. doi:10 1371/journal pone 0016724 g007 length intermediary between the Col and the fry1-6 PR lengths (Fig. S5B). In this mutant, the levels of the phosphate-starvation markers that appear induced in fry1 are comparable to the control levels (Fig. S4). In addition, the xrn4 single mutant is not drought tolerant (Estavillo and Pogson, personal communication). The PHT1;4:GUS induction in fry1 is unrelated to its action on XRNs Inter- estingly, the xrn2 xrn3 drought tolerance level is intermediary between the wild type and the fry1 drought tolerance levels (Estavillo and Pogson, personal communication), whereas the rosette phenotype of the double mutant is similar to that of the fry1-6 mutant [9]. Moreover, both fry1 mutants and the xrn2 xrn3 xrn4 triple mutant tolerate a drought stress that is lethal for the wild type controls (Fig. 7A), even though the rosette shape of the triple mutant is quite different from the fry1 rosette (compare the petiole length in fry1-6 and the xrn2 xrn3 xrn4 triple mutant in Fig. 7A). Thus, the drought tolerance observed in both fry1 and xrn2 xrn3 xrn4 triple mutants is not linked to a reduced leaf biomass Table 1. PHT1;4:GUS expression in different xrn backgrounds. Number of F2 plantlets stained/total nb of plantlets assayed Genetic cross on Pi depleted media on Pi complete media xrn2 X PHT1;4:GUS 16/24 0/24 xrn3 X PHT1;4:GUS 17/24 0/24 xrn4 X PHT1;4:GUS 34/45 0/204 xrn2 xrn4 X PHT1;4:GUS 39/58 0/474 xrn2 xrn3 xrn4 X PHT1;4:GUS 18/22 0/347 The F2 progeny of the indicated crosses were grown 7 to 10 days on either a complete or depleted Pi media before the GUS staining. Results on the Pi depleted media serve as a positive control for the presence of the PHT1;4:GUS transgene. Note that on a Pi-rich media, none of the seedlings expressed the GUS reporter gene. doi:10.1371/journal.pone.0016724.t001 Table 1. PHT1;4:GUS expression in different xrn backgrounds. The F2 progeny of the indicated crosses were grown 7 to 10 days on either a complete or depleted Pi media before the GUS staining. Results on the Pi depleted media serve as a positive control for the presence of the PHT1;4:GUS transgene. Note that on a Pi-rich media, none of the seedlings expressed the GUS reporter gene. doi:10.1371/journal.pone.0016724.t001 The F2 progeny of the indicated crosses were grown 7 to 10 days on either a complete or depleted Pi media before the GUS staining. Results on the Pi depleted media serve as a positive control for the presence of the PHT1;4:GUS transgene. Note that on a Pi-rich media, none of the seedlings expressed the GUS reporter gene. doi:10.1371/journal.pone.0016724.t001 February 2011 \| Volume 6 \| Issue 2 \| e16724 PLoS ONE \| www.plosone.org 8 FRY1 Activity Unlinked to XRNs Figure 8. Model accounting for the dual mode of action of FRY1. The PHT1;4:GUS induction in fry1 is unrelated to its action on XRNs The expression of FRY1 in the shoot is essential for root growth, drought resistance, and likely many other developmental aspects. This systemic mode of action relies on the XRN activities. By contrast, FRY1 has a local (i.e. not systemic) effect on the expression of the PHT1 genes and other phosphate starvation markers in roots; this effect depends on Pi accumulation but not on the XRN activities. doi:10.1371/journal.pone.0016724.g008 Figure 8. Model accounting for the dual mode of action of FRY1. The expression of FRY1 in the shoot is essential for root growth, drought resistance, and likely many other developmental aspects. This systemic mode of action relies on the XRN activities. By contrast, FRY1 has a local (i.e. not systemic) effect on the expression of the PHT1 genes and other phosphate starvation markers in roots; this effect depends on Pi accumulation but not on the XRN activities. doi:10.1371/journal.pone.0016724.g008 and transpiration, but rather to reduced XRN activity. All of the phenotypes linked to perturbations in XRN activities can be complemented by grafting, suggesting the presence of a systemic signal (Fig. 8, left). On the contrary, the induction of phosphate starvation markers is likely linked to a local effect of FRY1 expression (Fig 8, right). It is not complemented by a wild type scion and is not mimicked by the xrn mutations or linked to the root architecture phenotype. example, the BYPASS1 locus is required in Arabidopsis to prevent constitutive production of a root-derived graft-transmissible signal that is sufficient to inhibit leaf initiation, leaf expansion and shoot apical meristem activity [17]. We demonstrate here that FRY1 in shoots controls root development in Arabidopsis. We have identified a novel FRY1 function modulating the transcription of several Pi starvation markers in the root stele. This is the first fry1 mutant phenotype reported to be independent of XRN activities. Instead, it is likely depending on FRY1 impact on the root cytosolic Pi pool, in stele and pericycle cell layers. Interestingly, this phenotype is not complemented by a wild type scion and therefore acts locally. example, the BYPASS1 locus is required in Arabidopsis to prevent constitutive production of a root-derived graft-transmissible signal that is sufficient to inhibit leaf initiation, leaf expansion and shoot apical meristem activity [17]. We demonstrate here that FRY1 in shoots controls root development in Arabidopsis. We have identified a novel FRY1 function modulating the transcription of several Pi starvation markers in the root stele. Plant material and growth conditions Plant material and growth conditions The PHT1;4:GUS line (originally referred to as pht1;4-1 in [10]) was isolated from a T-DNA mutagenized A. thaliana ecotype Wassilewskija (Ws) seeds collection, obtained from INRA [18]. fry1-6 (SALK_020882), xrn2-1 (SALK_041148), xrn3-3 (SAIL_ 1172C07) and xrn4-6 (SALK_014209) mutants as well as the xrn2 xrn3, xrn2 xrn4 and xrn3 xrn4 double mutants have been described before [9]. Because XRN3 and XRN4 are genetically linked on chromosome 1, whereas XRN2 is on chromosome 5, the xrn2 xrn3 xrn4 triple mutant was generated by crossing xrn2 xrn4 to xrn3 xrn4 so that 1/16 of the F2 plants would be homozygous for the three mutations (xrn2 and xrn3 being genetically independent). The PHT1;4:GUS induction in fry1 is unrelated to its action on XRNs This is the first fry1 mutant phenotype reported to be independent of XRN activities. Instead, it is likely depending on FRY1 impact on the root cytosolic Pi pool, in stele and pericycle cell layers. Interestingly, this phenotype is not complemented by a wild type scion and therefore acts locally. We propose that this effect could be a result of the by-products of FRY1 activity, more specifically the result of the conversion of PAP into AMP + Pi. A reduction of FRY1 activity likely leads to a slight reduction of AMP and phosphate levels (along with an accumulation of PAP) in the tissues where FRY1 is normally very active (the root pericycle, central cylinder and meristems). The reduction of Pi availability would lead to the transcriptional induction of several phosphate starvation genes (including the PHT1;4:GUS marker) in these cell layers. This effect is not complemented by a wild type scion and is not mimicked by the xrn mutations or linked to the root architecture phenotype. February 2011 \| Volume 6 \| Issue 2 \| e16724 Mutant complementation and tissue localization of FRY1 Mutant complementation and tissue localization of FRY1 The FRY1 genomic fragment (1960 bp) and an additional 753 bp upstream region was PCR cloned by standard molecular techniques in the Ws accession. After sequencing in the pENTR/ D-TOPO (Invitrogen, Carlsbad, USA), an LR clonase (Invitrogen, Carlsbad, USA) reaction was used to clone the genomic fragment in the binary vector pGWB4 [25]. Then, the Arabidopsis fry1-7 mutant was transformed by a simplified floral dip method [26]. Similar construct were built with the FRY1 cDNA (1221 bp) with or without a C-terminal GFP fusion, under the control of the 35S promoter. Primary transformants were selected in medium containing 50 mg/L hygromycin. Their progeny was screened for root phenotype and GFP expression in standard in vitro growing conditions using a Leica SP2 AOBS inverted confocal microscope (Leica Microsystems, Germany) equipped with an Argon ion laser. Prior to confocal observation, plantlets were stained 3 min in 100 mg/mL propidium iodide (PI). Leaf shape, flowering time and GFP expression in mature plants were screened in soil-grown plants, both in short and long days conditions. To analyze mRNA splice variants, 10 mg of total RNA from roots and leaves were treated with DNase1 (Roche Diagnostics, Meylan, France) for 15 min at 37uC and were used for the reverse transcription reaction using the AMV Reverse Transcriptase (Roche Diagnostics, Meylan, France) according to the manufac- turer’s instructions. Specific primers (sequence available on request) were used to amplify FRY1 transcripts, both in the wild type and the mutant backgrounds. DNA cloning and sequencing were performed by standard procedures [31]. Mutagenesis, screening conditions and histology Approximately 3000 seeds of PHT1;4:GUS were mutagenized with a 0.3% solution of Ethyl methane sulfonate (EMS) as described [20]. Seeds were sown and the M1 plants were cultivated to obtain the M2 generation. Around 30 seeds of each M2 line (1400 lines) were sown in 6-well Petri dishes (NUNC) containing a modified Hoagland medium (1 mM MgSO4, 2 mM Ca(NO3)2, 1.7 mM KNO3, 1.6 mM Fe, 46.2 mM H3BO3, 9.1 mM MnCl2, 0.87 mM ZnSO4, 0.32 mM CuSO4, 1.03 mM Na2MoO4, 0.5 mM NH4H2PO4). After 10 days, seedlings were screened for their GUS expression as described [10]. Histological analysis were performed as described [21]. Grafting experiments y p g The mutant line was backcrossed three times to the parental line (PHT1;4:GUS) to test the linkage of the phenotype to a single Mendelian recessive mutation. For mapping purposes, a mutant plant (Ws ecotype) was crossed with a wild-type Col plant. Linkage analysis was performed with the F2 progeny of this cross as described [22]. DNA from F2 seedlings displaying the mutant phenotypes (GUS staining of a root piece from seedlings grown on complete media) was prepared as described [23]. Single Sequence Length Polymorphism markers [24] distributed on the five chromosomes and polymorphic between the Ws and Col accessions were tested on the extracted DNA. Thermal cycling consisted of an initial denaturation at 94uC for 2 minutes, followed by 38 cycles of denaturation step at 94uC for 15 seconds, annealing at the respective Tm of each oligonucleotide pair for 20 seconds, and extension at 72uC for 45 sec. At the end of the reactions, the PCR products were allowed to extend for 2 minutes at 72uC. Grafting was performed according to [30]. Parental and mutant lines were sown in vitro on a MS/10 medium. Four days after sowing they were cut at the hypocotyl level to separate the aerial and root parts. A 0.3 mm diameter silicon ring (Silastic Laboratory tubing, Dow Corning, USA) was used to maintain the aerial seedling scion and the rootstock together to allow fusion. After five days, successful grafts were transferred to fresh medium for 48 hours, followed by GUS staining for 16 h at 37uC. Alternatively, established grafts were put on soil, either on large soil-filled plates or in pots and grown in the greenhouse for 4 weeks in order to assess the root architecture and the drought tolerance of the grafts. For drought tolerance, plants were either cultured in long days (12 h light, 12 h dark), before watering was withheld, then survival of the plants was determined as described [19] or cultured in short days (8 h light, 14 h dark) using a mix of J soil and L sand and an immersion watering per day. Phenotype was assessed after 13 days without watering followed by three days were watering of the individual pots was resumed. To identify the mutant locus on chromosome 5, the Gramene Simple Sequence Repeat Identification Tool (SSRIT, http:// www.gramene.org/db/markers/ssrtool) was used to generate new markers in the area surrounding FRY1. Conclusion Long-distance signaling is used by plants to coordinate shoot and root development. Despite the importance of such coordina- tion, only a few genes have been shown to regulate root development in a systemic way. For example, in Lotus japonicus, the use of reciprocal and self-grafting studies with the hyperno- dulating mutant har1 have shown that the shoot genotype is responsible for the negative regulation of nodule development. Therefore, HAR1 in shoots mediates systemic regulation of nodulation [16]. There are also few examples of root genes regulating shoot development by long-distance signaling. For For physiological analyses and RNA extractions, seeds of Col-0, Ws, PHT1;4:GUS, and the different fry1 mutant alleles were cultivated as described before [10]. For drought tolerance tests, plants were grown in individual pots in short days for 4 weeks with standard watering conditions (once a day). Watering was stopped PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16724 February 2011 \| Volume 6 \| Issue 2 \| e16724 9 FRY1 Activity Unlinked to XRNs for 13 days and the pots were then rehydrated for 3 days before the observations. Alternatively, after the onset of wilting, survival of the plants was quantified by measuring chlorophyll fluorescence as described [2,19]. software (http://rsb.info.nih.gov/ij/). To determine the speed of growth of the main root, photographs were taken at 8, 11 and 14 days post germination (dpg) from which PR length was measured. The daily growth was calculated accordingly. To measure single cell length above the differentiation zone, roots were briefly stained with ruthenium red and observed with a bright field microscope (Leica DMRXA, 20x objective). At least 30 cells for each of 12 different roots per genotype were measured using a micrometric lens. To estimate the size of the proximal meristem (PM), the number of undifferentiated cells in the cortex was measured in at least 30 roots per genotype as described [27,28]. PI-stained roots (3 min in 100 mM PI) were observed by confocal laser scanning microscopy. PI was excited at 514 nm and imaged using a custom 610–720 nm band pass emission filter. To determine the number of LR primordia at different stages of development, we used a Nomarsky optical microscope, as described [29]. All experiments were performed at least three times. Molecular and gene expression analysis For gene expression analysis, total RNA was extracted from rosettes and roots of 10 day-old plantlets of the PHT1;4:GUS parental line and the PHT1;4:GUS/fry1-7 mutant, grown in MS/ 10 medium as described previously [12]. cRNA was prepared using the manufacturer’s instructions (www.affymetrix.com sup- port technical manual expression_manual.affx). Labeling and hybridization on the ATH1 microarray and data analysis were performed according to [12]. Microarray data has been deposited at the EMBL database with the accession number E-MEXP-2483 (www.ebi.ac.uk/arrayexpress) and was used in the present work to identify candidate genes during the positional cloning of the mutant locus. Analysis of root architecture RTqPCR analyses were performed after reverse transcription (kit from GE Healthcare) and amplification (Applied ABI7000). Primer efficiency factors were measured for each gene and GapC Seedlings were photographed at different times after germina- tion and PR and LR length were measured with the ImageJ PLoS ONE \| www.plosone.org February 2011 \| Volume 6 \| Issue 2 \| e16724 February 2011 \| Volume 6 \| Issue 2 \| e16724 10 FRY1 Activity Unlinked to XRNs and ROC3 were used as reference genes. Primer sequences are available upon request. Figure S3 Expression of phosphate-starvation induced genes in the 35S:AHL complemented line. (A) Quantitative real time PCR of the PHT1;4 transcripts in Col, fry1-6 and fry1-6/ 35S::AHL roots. (B) Quantitative real time PCR on the Pho1H1 transcript in Col, fry1-6 and fry1-6/35S::AHL roots. (C) Quanti- tative real time PCR on the At1g73010 phosphatase transcript in Col, fry1-6 and fry1-6/35S::AHL roots. Biological triplicates were performed and all samples were analyzed with technical triplicates. White bars correspond to Col roots, grey bars to fry1-6 roots and black bars to fry1-6/35S::AHL roots. Standard deviations are shown. (EPS) Supporting Information Figure S1 Mutant complementation assays and leaf phenotype of PHT1;4:GUS/fry1-7 mutant. (A) Complemen- tation of the fry1-7 mutant. The progeny of a plant heterozygous for a T-DNA carrying a 35S::FRY1 cDNA construct is shown. Asterisks indicate non-complemented fry1-7 mutant plantlets that, presumably, did not inherit the transgene. (B) Picture of the rosette of the 3 week-old PHT1;4:GUS line (left) and the PHT1;4:GUS/ fry1-7 mutant (right) grown on soil under short day conditions. (C) 6 week-old PHT1;4:GUS line (left) and the PHT1;4:GUS/fry1-7 mutant (right) grown in long day conditions. (D) Complementation of the fry1-6 mutant with a 35S::AHL cDNA. The Col control (left), the fry1-6 mutant (middle) and the complemented line (fry1-6/ 35S::AHL) (right) are shown. White scale bars are 20 mm. (EPS) Figure S5 Root development and primary root length of the xrn4-6 mutant. (A) The general in vitro development of Col, xrn4-6 and fry1-6 mutants 11 dpg. Scale bars are 20 mm. (B) PR length was measured at 11 dpg. White bars correspond to Col, grey bars to fry1-6 and black bars to xrn4-6 mutant. Standard deviations are shown. (EPS) Production of Arabidopsis transformants expressing pAT1G73010::LUC A DNA fragment corresponding to 2001 bp of the promoter driving the expression of the AT1G73010 gene (ending right before the ATG) was PCR amplified and cloned into the pENTER-D-TOPO vector. The fragment was recombined into the pBGWL7 vector [32] using LR clonase. After sequencing confirmation, the vector was introduced into C58C1 Agrobacterium tumefaciens cells. Arabidopsis plants were transformed using a modified floral dip method [26], and transformed plants were selected using Basta (T1). Figure S4 Expression of phosphate-starvation induced genes in the fry1-6, xrn4 and xrn2 xrn3 xrn4 mutants. Quantitative real time PCR of the PHT1;4 transcript (A), the PHT1,7 transcript (B), the Pho1H1 transcript (C) and the At1g34010 phosphatase transcript (D) in Col, fry1-6, xrn4-6 and xrn2 xrn3 xrn4 roots. Biological triplicates were performed and all samples were analyzed with technical triplicates. White bars correspond to Col, pale grey to fry1-6, dark grey to xrn4-6 and black bars to xrn2 xrn3 xrn4 mutant. Standard deviations are shown. Bioluminescence detection was performed on the T2 generation (8 day-old plantlets) using a UPLSAPO 4X dry objective (N.A. 0.16) or a LUCPLFLN 40X dry objective (N.A. 0.6) mounted on an Olympus LV200 Luminoview microscope coupled to an ANDOR iKon-M DU934 camera. Images were acquired with an exposure time of 2 min (4X objective) or 4 min (40X objective). Contrast and brightness of the images were adjusted in ImageJ. Bioluminescence detection was performed on the T2 generation (8 day-old plantlets) using a UPLSAPO 4X dry objective (N.A. 0.16) or a LUCPLFLN 40X dry objective (N.A. 0.6) mounted on an Olympus LV200 Luminoview microscope coupled to an ANDOR iKon-M DU934 camera. Images were acquired with an exposure time of 2 min (4X objective) or 4 min (40X objective). Contrast and brightness of the images were adjusted in ImageJ. Acknowledgments The authors are very grateful to Drs B.-H. Kim and A.G. von Arnim (University of Tennessee, USA) who provided seeds of their transgenic lines and to Dr. B. Albaud for performing array hybridizations (Affymetrix platform, Curie Institut, Paris, France). We acknowledge TAIR (http:// arabidopsis.org) as a source of data and the Arabidopsis Biological Resource Center (ABRC) for the fry1-6 mutant. We are grateful to Nathalie Pochon for technical help and to Dr. Brandon Loveall for English proofreading of the manuscript. Figure S2 The pAT1G73010::LUC construct reveals the stele specificity of gene expression in Arabidopsis roots and the phosphate starvation induction. (A) Transmitted light image and (B) bioluminescence signal of pAT1G73010::LUC plantlets grown for 4 days on P depleted medium then for 4 days on complete medium (plantlet on the left) or for 8 days on P depleted medium (plantlet on the right). Scale bar, 1 mm. (C) Close up of a mature part of a root from a plantlet grown for 8 days on P depleted medium (overlay of transmitted light and bioluminescence signal). The bioluminescence signal is only detected in the central cylinder. Scale bar, 100 mM. (EPS) Author Contributions Conceived and designed the experiments: JH GME BJP LN EM. Performed the experiments: JH JM PAC PD VB HJ SC ACM MD HV EM. Analyzed the data: JH JM PAC PD ACM AM HV MC TD MCT LN EM. Contributed reagents/materials/analysis tools: JM ACM AM EM. Wrote the paper: MC TD LN EM. 7. Gil-Mascarell R, Lopez-Coronado JM, Belles JM, Serrano R, Rodriguez PL (1999) The Arabidopsis HAL2-like gene family includes a novel sodium-sensitive phosphatase. Plant J 17: 373–383. 6. Quintero FJ, Garciadeblas B, Rodriguez-Navarro A (1996) The SAL1 gene of Arabidopsis, encoding an enzyme with 39 (29),59-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities, increases salt tolerance in yeast. Plant Cell 8: 529–537. 8. 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W2969592920.txt	null	fr		Syndrome de la selle turcique vide: à propos d’un cas	The Pan African medical journal	2,019	cc-by	1,735	Open Access Case report Syndrome de la selle turcique vide: à propos d’un cas Empty sella syndrome: a case report Benilde Bepouka Izizag1,2,&, Aaron Ngandu 1, Daddy Liombo Mbiso3 1 Service de Médecine Interne, Clinique Rapha, Kinshasa, République Démocratique du Congo, 2Département de Médecine Interne, Cliniques Universitaires de Kinshasa, Kinshasa, République Démocratique du Congo, 3Service d’Imagerie Médicale, Cliniques Universitaires de Kinshasa, Kinshasa, République Démocratique du Congo & Auteur correspondant: Benilde Bepouka Izizag, Service de Médecine Interne, Clinique Rapha, Kinshasa, République Démocratique du Congo Mots clés: Syndrome, selle turcique, vide Received: 22/10/2018 - Accepted: 28/01/2019 - Published: 21/08/2019 Résumé Le syndrome de la selle vide est une affection dans laquelle la selle turcique est partiellement ou complètement remplie de liquide céphalorachidien (LCR), entraînant un déplacement de l'hypophyse. Nous rapportons le cas d'un patient obèse de 49 ans qui a présenté de façon progressive les céphalées, une asthénie physique et une hypothyroïdie et chez qui le scanner cérébral a été en faveur d'un syndrome de la sel le turcique vide. Pan African Medical Journal. 2019;33:317. doi:10.11604/pamj.2019.33.317.17423 This article is available online at: http://www.panafrican-med-journal.com/content/article/33/317/full/ © Benilde Bepouka Izizag et al. The Pan African Medical Journal - ISSN 1937-8688. 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. Pan African Medical Journal – ISSN: 1937- 8688 (www.panafrican-med-journal.com) Published in partnership with the African Field Epidemiology Network (AFENET). (www.afenet.net) Page number not for citation purposes 1 Abstract Empty sella syndrome is a condition in which the sella turcica is partially or totally filled with cerebrospinal fluid causing a displacement of the pituitary gland. We here report the case of a 49-year old obese patient with progressive headaches, physical asthenia and hypothyroidism. Brain scanner showed empty sella syndrome. Key words: Syndrome, sella turcica, empty Introduction Discussion Le syndrome de la selle turcique vide (SSTV) est un trouble impliquant La selle turcique vide (STV) est une hernie à travers le diaphragme la selle turcique qui est une structure osseuse située à la base du sellaire de l'arachnoïde suivie de l'espace sous-arachnoïdien cerveau et entourant la glande pituitaire [1]. Le syndrome de la selle supra-sellaire, rempli de liquide céphalorachidien, dans la selle vide est une affection dans laquelle la selle turcique est partiellement turcique [3]. Le terme STV est incorrect car en réalité la selle turcique ou complètement remplie de liquide céphalorachidien (LCR), n'est pas vide. Elle est plutôt complètement rempli par l'hypophyse, entraînant un déplacement de l'hypophyse [2]. Nous rapportons le sa tige, l’arachnoïde, le LCR et parfois le système optique et le cas d'un patient qui présente une hypothyroïdie révélant un syndrome troisième ventricule. C'est pourquoi certains auteurs préfèrent utiliser de la selle turcique. le terme arachnoidocèle intrasellaire car il exprime clairement cette entité [4]. Il existe deux types de SSTV: primaire et secondaire. Le SSTV primaire survient suite à un petit défaut anatomique au-dessus de l'hypophyse qui augmente la pression dans la selle turcique et Patient et observation provoque l’aplatissement de la glande le long des parois intérieures Patient âgé de 49 ans qui consulte pour céphalées, des troubles visuels sous forme de vision floue, une asthénie physique et une sensation des crampes à l'hémicorps gauche évoluant depuis un mois. Dans ses antécédents nous notons qu'il est marié et père de 4 enfants. A l'examen physique, le patient a un bon état général, une pression artérielle à 122/72 mmHg, une fréquence cardiaque à 64 battements par minute, un indice de masse corporel à 30 kg/m2; le reste de l'examen est normal. Le bilan ophtalmologique est normal. L'échographie cardiaque, l'électrocardiogramme (ECG) et la radiographie du thorax sont normaux. Les explorations de laboratoire incluant l'hémogramme, la fonction rénale, la fonction hépatique, les électrolytes sont dans les normes. L'exploration hormonale révèle une hypothyroïdie tandis que les gonadotrophines, le cortisol, la prolactine et l'hormone de croissance sont dans les normes. L'image tomodensitométrique a révélé une vacuité de la loge de la selle turcique avec petitesse des ventricules latéraux, l'absence de toute lésion malformative de la ligne médiane, du cortex ni de la fosse postérieure (Figure 1). Le traitement de substitution à la L-Thyroxine (50µg/l) entraine la disparition de l'asthénie physique et des crampes de l'hémicorps gauche. Bilan hormonal: TSH: 7,45 (VN: 0,55-4,78); T3: 3.07 pg/ml (2.30-4.20); T4: 0.75 pg/ml (0.93-1.70); ACTH: 53,09 (VN: 7.20-63.3); LH: 4,78 mUI /ml (VN: 4.04-15.20); Prolactine: 10.40 (VN: 4.04-15.20); GH: 0.032 ng /ml (VN: 0.030-2.47). de la cavité de la selle turcique. Le syndrome primaire se rencontre généralement chez les femmes d’âge moyen, obèses et hypertendues. Le trouble peut être un signe d’hypertension intracrânienne idiopathique [5]. Parmi les facteurs de risque il y a l'obésité chez la femme et la multiparité. L'obésité entraine l'apnée du sommeil obstructive avec une hypercapnie qui peut augmenter la pression du LCR et prédisposer au SSTV [6]. Ceci peut être le cas de notre patient bien qu'il soit de sexe masculin. Le SSTV secondaire résulte de la régression de l'hypophyse dans la cavité après une blessure, une chirurgie ou une radiothérapie. Les personnes atteintes de SSTV secondaire en raison de la destruction de l’hypophyse présentent des symptômes qui traduisent la perte des fonctions hypophysaires, telles que la cessation des menstruations, la stérilité, la fatigue et l’intolérance au stress et aux infections [7]. La selle turcique vide peut se traduire par plusieurs signes cliniques. Les céphalées, classiquement fronto-orbitaires, sont présentes dans 50% des cas, mais la responsabilité de la selle turcique vide est discutée par de nombreux auteurs [6-8]. Notre patient présentait aussi des céphalées frontales. La fréquence des troubles visuels varie selon les séries mais semble peu élevée: l'altération du champ visuel (hémianopsie bitemporale voire quadranopsie bitemporale supérieure) est le symptôme le plus Page number not for citation purposes 2 fréquent; un œdème papillaire voire une atrophie optique sont plus Conclusion exceptionnels [8]. Notre patient présentait un flou visuel mais l'examen ophtalmologique n'a rien révélé de particulier. Les signes endocriniens ne sont pas exceptionnels et peuvent se présenter sous forme de syndrome d'hyposécrétion hormonale ou d'hypersécrétion hormonale. Le syndrome d'hyposécrétion hormonale ou hypopituitarisme, souvent partiel est retrouvé dans 15 à 30% des syndromes de selles turciques vides primaires. Parfois, les tests Le SSTV est arachnoidocèle intrasellaire souvent de découverte fortuite caractérisé par une élévation transitoire ou constante de la pression intracrânienne. Il est souvent rencontré chez les obèses. Il doit être considéré comme diagnostic différentiel chez les patients ayant des maux de tête non spécifiques, en particulier les obèses. hormonaux dynamiques peuvent être discrètement perturbés: absence de stimulation de la fonction thyréotrope ou corticotrope ou, le plus souvent, somatotrope. Le pan hypopituitarisme est très Conflits d’intérêts rare [6]. Dans notre cas, le patient avait une insuffisance thyréotrope bien qu'il n'avait pas de signe clinique d'hypothyroïdie. Cette Les auteurs ne déclarent aucun conflit d'intérêts. hypothyroïdie apparait fortuitement dans un contexte de SSTV probablement primaire et a bien évolué après substitution hormonale. Dans le syndrome d'hypersécrétion hormonale, la manifestation la Contributions des auteurs plus fréquente est l'hyperprolactinémie qui peut être due à un micro adénome et/ou à l'interruption du frein dopaminergique hypothalamique [9]. Du point de vue du diagnostic en imagerie Tous les auteurs ont lu et approuvé la version finale du manuscrit. médicale, la radiographie latérale du crâne peut révéler une selle de taille normale ou être agrandi. La selle vide typique montre une «montgolfière symétrique», c'est-à-dire une cavité régulière et Figure incurvée de la selle. Actuellement, elle est devenue obsolète et insuffisante. Des tomographies informatisées montreront que la fosse Figure 1: TDM cérébrale en fenêtre parenchymateuse coupes pituitaire est occupée en grande partie par la substance du LCR ou la sagittale (A) et coronale (B): présence d’une hypodensité dans la loge densité de l'eau plutôt que par une glande normale. L'imagerie par sellaire ressemblant à l’hypodensité du 3 ème ventricule, le tissu résonance magnétique (IRM), méthode de choix dans l'exploration de hypophysaire résiduel plaqué contre le plancher sellaire traduisant la région hypophysaire, peut facilement confirmer le diagnostic d’un donc la présence du LCR dans la loge: SSTV sella vide. Sur les images IRM sagittales T1, l'extension du LCR dans la selle est facilement identifiable et la glande restante est comprimée le long du sol. La position centrale typique de l’infundibulum est un signe utile d'une selle vide qui permet d’éliminer une lésion kystique dans la région supra-sellaire. L’IRM démontrera que la sella est remplie de LCR et que l'infundibulum traverse l'espace, excluant ainsi Références 1. syndrome: a case report. AKMMC J. 2010; 1(1):23-25. Google une masse kystique. C'est ce que l’on appelle le signe de Scholar l'infundibulum [7]. L'accessibilité à l'IRM étant encore limitée à cause de sa non disponibilité mais aussi et surtout à cause de son cout élevé dans nos milieux, nous avons posé notre diagnostic par le scanner Hossain MS, Mumu MA, Moyenuddin PK. Primary empty sella 2. Wael Fouad. Review of empty sella syndrome and its surgical management. Alexandria Journal of Medicine. 2011; 47: 139- cérébral. 147. Google Scholar 3. Agrawal JK, Sahay RK, Bhutada SK, Reddy VS, Agrawal NK. Empty sella syndrome. Indian Academy of Clinical Medicine. 2001; 2(3):198-02. Page number not for citation purposes 3 4. 5. Sander EC, Peter Jr WC. Empty sella syndrome In: Wilkins R, 7. Deshpande PK, Deoke S, Yesankar V, Banait A, Sawarkar S, Rengachery S, editors. Neurosurgery, New York: McGraw- Dhande S. Empty sella syndrome: a case report. Panacea J Med Hill.1996; 1367-73. Sci. 2015;5(1):52-3. Jordan RM, Kendall JW, Kerber CW. The primary empty sella 8. Braatvedt GD, Corral RJM. The empty sella syndrome: much ado syndrome: analysis of the clinical characteristics, radiographic about nothing. Br J Hosp Med. 1992; 47: 523-5. PubMed \| features, Google Scholar pituitary function and cerebrospinal fluid adenohypophysial hormone concentrations. Am J Med. 1977; 62:569. Google Scholar 9. Robinson Douglas B, Michaels Rodney D. Empty sella resulting from the spontaneous resolution of a pituitary macroadenoma. 6. De Marinis L, Bonadonna S, Bianchi A, Maira G, Giustina A. Arch Intern Med. 1992; 152 : 1920-3. PubMed \| Google Primary empty sella. J Clin Endocrinol Metab. 2005;90:5471-7. Scholar PubMed \| Google Scholar Figure 1: TDM cérébrale en fenêtre parenchymateuse coupes sagittale (A) et coronale (B): présence d’une hypodensité dans la loge sellaire ressemblant à l’hypodensité du 3ème ventricule, le tissu hypophysaire résiduel plaqué contre le plancher sellaire traduisant donc la présence du LCR dans la loge: SSTV Page number not for citation purposes 4
https://openalex.org/W2973884986	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0222166&type=printable	English	null	Ocular and systemic risk factors associated with recurrent disc hemorrhage in primary open-angle glaucoma	PloS one	2,019	cc-by	7,202	RESEARCH ARTICLE Bo Ram Seol1,2, Jin Wook Jeoung2, Ki Ho ParkID2* 1 Department of Ophthalmology, VHS Medical Center, Seoul, Korea, 2 Department of Ophthalmology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea 1 Department of Ophthalmology, VHS Medical Center, Seoul, Korea, 2 Department of Ophthalmology, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea * kihopark@snu.ac.kr * kihopark@snu.ac.kr * kihopark@snu.ac.kr Editor: Andrzej Grzybowski, University of Warmia, POLAND Received: April 8, 2019 Accepted: August 22, 2019 Published: September 16, 2019 Purpose To evaluate the risk factors associated with recurrent disc hemorrhage (DH), defined for the present study as at least 3 occurrences of DH in primary open-angle glaucoma (POAG). Abstract a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS A total of 178 POAG patients (89 eyes showing at least 3 occurrences of DH and 89 age- matched control eyes with a minimum of 10 years’ follow-up without DH) were included in a retrospective, case-control study. Ocular factors were evaluated by a retrospective chart review, and systemic factors were evaluated by a telephone survey. Associations between factors and recurrent DH were investigated by logistic regression analysis. The Kaplan– Meier survival analysis and Cox proportional-hazards regression models were used to evaluate glaucoma progression and to identify the factors predictive of glaucoma progression. Citation: Seol BR, Jeoung JW, Park KH (2019) Ocular and systemic risk factors associated with recurrent disc hemorrhage in primary open-angle glaucoma. PLoS ONE 14(9): e0222166. https://doi. org/10.1371/journal.pone.0222166 Editor: Andrzej Grzybowski, University of Warmia, POLAND Ocular and systemic risk factors associated with recurrent disc hemorrhage in primary open-angle glaucoma Bo Ram Seol1,2, Jin Wook Jeoung2, Ki Ho ParkID2* Results Copyright: © 2019 Seol 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. Univariate regression analysis revealed the association of recurrent DH with low baseline intraocular pressure (IOP) [odds ratio (OR), 0.88; 95% confidential interval (CI), 0.80–0.98; P = 0.014], lower percentage reduction of IOP (OR, 0.96; 95% CI, 0.93–0.99; P = 0.020), cold extremities (OR, 2.80; 95% CI, 1.03–7.60; P = 0.043), prone or lateral decubitus sleep- ing position (OR, 2.14; 95% CI, 1.13–4.03; P = 0.019), and sleeping disorders (OR, 2.33; 95% CI, 1.05–5.15; P = 0.037). Multivariate regression analysis revealed that a lower per- centage reduction in IOP (OR, 0.96; 95% CI, 0.93–1.00; P = 0.046) increased the risk of recurrent DH. The control group exhibited a greater cumulative probability of non-progres- sion than the recurrent DH group (P = 0.01, by log-rank test). The Cox proportional-hazards regression model showed that recurrent DH was associated with glaucoma progression [hazard ratio (HR), 1.88; 95% CI; 1.66–3.05; P = 0.01. Conclusions Among the ocular and systemic factors, only lower-percentage reduction of IOP in POAG was associated with recurrent DH. DH recurrence is associated with glaucoma progression and may be dependent on IOP. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. 1 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0222166 September 16, 2019 Risk factors associated with recurrent disc hemorrhage Introduction Previous studies have reported disc hemorrhage (DH) as an important risk factor for the devel- opment and progression of glaucoma [1, 2]. The Ocular Hypertension Treatment Study dem- onstrated that DH is a specific risk factor for the development of primary open-angle glaucoma (POAG), while the Early Manifest Glaucoma Trial, showed a significant association between DH and visual field (VF) progression [3, 4]. However, DH can occur not only in eyes with glaucoma but also in normal eyes. For example, the Blue Mountain Eye Study noted the presence of DH not only in 13.8% of participants with open-angle glaucoma (8% of patients with high-pressure glaucoma and 25% with low-pressure glaucoma) and 1.5% of patients with ocular hypertension but also in 1% of normal eyes [5]. Based on the possible occurrence of DH in normal eyes, we surmised that an evaluation of recurrent DH might be more appropriate, and certainly important, for glaucoma patients. Although the recurrence of DH is common, there are few studies that have investigated it. Those that have focused on the relationship between recurrent DH and glaucoma progression and reported controversial results [6–8]. Siegner et al.[6] and de Beaufort et al.[7] noted that there were no differences in the rates of glaucoma progression between recurrent-DH patients and single-DH patients. However, Ishida et al. [8] reported that recurrent DH eyes exhibited a more pronounced glaucoma progression than did single-DH eyes. In consideration of this cor- relation between recurrent DH and glaucoma progression, we considered an investigation into the risk factors associated with DH recurrence was necessary, as such knowledge could contribute to our understanding of the causative mechanisms behind it. To the best of our knowledge, no studies on the risk factors of recurrent DH has yet to be conducted. Therefore, we evaluated the risk factors associated with DH recurrence in POAG patients. PLOS ONE \| https://doi.org/10.1371/journal.pone.0222166 September 16, 2019 Subjects We conducted a retrospective chart review of POAG patients who had visited the glaucoma clinic at Seoul National University Hospital from January 2002 through March 2016. The study protocol, approved by the Institutional Review Board (IRB) of Seoul National University Hospital, adhered to the tenets of the Declaration of Helsinki. Due to the retrospective data analysis, the ethics committee waived off the requirement of informed consent. In addition, the IRB approved the use of oral consent for evaluating systemic factors by telephone survey. One glaucoma specialist (B.R.S.) explained the purpose of the survey to the participants, all of whom provided their oral consent. All of the patients underwent a complete ophthalmologic examination including visual acu- ity (VA), intraocular pressure (IOP) measurement (Goldmann applanation tonometry), cor- neal pachymetry (Pocket II Pachymeter Echo Graph; Quantel Medical, Clermont-Ferrand, France), measurement of axial length (AXL; Axis II PR, Quantel Medical, Inc., Bozeman, MT, USA), slit-lamp examination, gonioscopy, Humphrey Visual Field Analysis (Carl Zeiss 2 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0222166 September 16, 2019 Risk factors associated with recurrent disc hemorrhage Meditec, Inc., Dublin, CA, USA) using the Swedish interactive threshold algorithm with a 30– 2 standard program, and spectral-domain optical coherence tomography (SD-OCT) (Carl Zeiss Meditec, Inc.). Color disc stereo photography and red-free fundus photography were evaluated at intervals of 3–6 months, and VF tests at intervals of 6–12 months, according to patient status. Participants who met the following criteria were included: (1) an open-angle glaucoma (OAG) diagnosis in 1 or both eyes at the first clinic visit, (2) no history of IOP-lowering treat- ment at baseline, and (3) treated with topical medications during the follow-up period. In addition, patients had to be older than 18 years of age and to have had a best-corrected visual acuity (BCVA) of 20/40 or better, good quality red-free photographs, and a reliable VF. Patients were excluded for any of the following reasons: retinal diseases that might affect the VF or the interpretation of red-free fundus photographs, including diabetic retinopathy and any history of other optic neuropathies. We further excluded those who had undergone glau- coma laser treatment (argon laser trabeculoplasty or selective laser trabeculoplasty) or glau- coma surgery (trabeculectomy or Ahmed valve operation). If both eyes were eligible for inclusion, one eye was selected randomly. Subjects All of the patients were treated for glaucoma at the discretion of the attending ophthalmologist (K.H.P.), who aimed to reduce baseline IOP by at least 20%. A diagnosis of POAG was made in cases where a patient showed glaucomatous optic disc damage and corresponding glaucomatous VF defects, along with an open angle. Glaucoma- tous optic disc changes were defined on stereoscopic color disc photography as cup/disc (C/D) asymmetry between the glaucomatous and normal eye that was greater than 0.2, large cupping (>0.7 vertical C/D ratio), and neuroretinal rim thinning, notching, or excavation. Only reli- able VF tests (false-positive errors < 15%, false-negative errors < 15%, fixation loss <20%) were included in the analysis. Optical Coherence Tomography (OCT) is widely used in clinical practice; however, various parameters can be used to diagnose glaucoma with OCT. For exam- ple, quadrant retinal-nerve fiber layer (RNFL) thickness, clock-hour RNFL thickness, ganglion cell-inner plexiform layer (GCIPL) thickness, the RNFL deviation map, and the GCIPL devia- tion map can all be used. As the purpose of this study was not to diagnose glaucoma, we con- sidered it sufficient to diagnose glaucoma using disc photography, RNFL photography, and the VF test. A narrow definition of recurrent DH was used, i.e., an eye showing at least 3 occurrences of DH during the follow-up period, with an interval between DH onsets of at least 6 months. Patients with eyes not showing any DH over the course of at least 10 years of follow-up were enrolled as part of the control group. The presence of DH was evaluated independently by 2 glaucoma specialists (B.R.S. and K.H.P.) and discrepancies between the two observers were resolved by consensus. Recurrent DH diagnosis was based on color disc stereo and red-free fundus photography and was defined as an isolated hemorrhage observed either in the optic disc or in the peripapillary retina and extending to the disc rim. Once the recurrent DH group was defined using the above definition, the control group was selected by age-matching. If DH was detected, we re-examined the patient several months later to confirm that it had disappeared. We did not change the treatment based on a one-time occurrence of DH. In cases where accompanying glaucoma progression was observed, or recurrent DH occurred, we escalated the treatment. Systemic factors Systemic factors were evaluated by telephone survey. This was done at the end of the follow-up period, and a standard phone script was used. The following systemic factors were investi- gated: diabetes, systemic hypertension, cardiac disease (ischemic heart disease or arrhythmia), the use of aspirin, anticoagulant and gingko extract, history of ocular operations and trauma, cold extremities, migraine, glaucoma family history, sleeping position (prone/lateral decubitus positions), yoga, sleep disorders (sleep apnea/snoring), constipation, carrying heavy loads, and wind-instrument playing. In the case of sleep apnea, self-reported snoring alone was not enough to confirm its presence, hence patients were asked if they had received a diagnosis of snoring. Constipation was defined as habitual constipation. Patients were checked for carrying heavy physical loads only if it was frequent and habitual. Wind-instrument playing was defined as that which required significant Valsalva maneuvers. We did not perform detailed patient assessments on the status and severity of these systemic diseases, and patients who did not receive treatment after a diagnosis were excluded. Patients were also excluded if they had been previously treated for one of these diseases and normalized without any need for further treatment. In addition, any participants who were unsure of, or could not remember their status regarding any of these systemic factors, or experienced a sig- nificant change in their systemic disease status were excluded from the study. There is always the possibility that a patient neglects to mention or cannot remember what is listed on their medical record. Therefore, we also conducted a medical chart review on of all of the items included in the telephone survey. Ocular factors were judged by prioritizing hospital records. However, in the case of systematic factors, if the patient received treatment at our hospital and the patient’s memory was different from the medical record, the hospital records were consid- ered to be correct. Conversely, a patient’s memory was considered correct if it differed from their medical records when they had been treated at other hospitals. Ocular factors We evaluated ocular factors by retrospective chart review. These factors included central cor- neal thickness (CCT), AXL, baseline IOP, IOP with medication (mean, fluctuation and 3 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0222166 September 16, 2019 Risk factors associated with recurrent disc hemorrhage maximum IOP, percent reduction of IOP), number of medications, VF global indices, and average RNFL thickness as measured by SD-OCT. Baseline IOP was measured prior to treat- ment and on every visit thereafter. Fluctuations of IOP was defined as the standard deviation during follow-up. The percent reduction of IOP was defined as follows: 100 (baseline IOP– mean IOP during treatment) / baseline IOP. maximum IOP, percent reduction of IOP), number of medications, VF global indices, and average RNFL thickness as measured by SD-OCT. Baseline IOP was measured prior to treat- ment and on every visit thereafter. Fluctuations of IOP was defined as the standard deviation during follow-up. The percent reduction of IOP was defined as follows: 100 (baseline IOP– mean IOP during treatment) / baseline IOP. PLOS ONE \| https://doi.org/10.1371/journal.pone.0222166 September 16, 2019 Statistical analysis Comparisons of the baseline characteristics between the two groups were performed using the independent t-test for continuous variables and the chi-squared test for categorical variables. Univariate and multivariate logistic regression analyses with backward stepwise selection were used to identify the risk factors for recurrent DH. Variables with P < 0.10 in the univariate analysis, were used for multivariate analysis. The intergroup cumulative risk ratios of glau- coma progression were compared using the Kaplan-Meier survival analysis and log rank tests. The detection of initial progression was regarded as the end point in the survival analysis. The end of follow-up was determined as the point at which patients without progression were cen- sored. The univariate and multivariate Cox proportional-hazards regression models with back- ward stepwise selection were used to identify risk factors for glaucoma progression. Variables with P < 0.10 in the univariate analysis were used for multivariate analysis. The proportional- hazards assumption was tested with the Schoenfeld residuals method and the assumption was not violated. Multicollinearity between factors was assessed by calculating the relevant variance inflation factors (VIF), to prevent multicollinearity. All the statistical analyses were performed using SPSS 18.0 (SPSS, Inc. Chicago, IL, USG) and R 3.5.1 (R Foundation, Vienna, Austria); P values less than 0.05 were considered statistically significant. Glaucoma progression Structural progression was determined by evaluating color disc and red-free RNFL photo- graphs. Progressive optic disc changes (e.g., focal or diffuse rim narrowing, neuroretinal rim notching, increased cup-to-disc ratios, and adjacent vasculature position shifts) were deter- mined by comparing serial disc photographs and regarded as indicating glaucoma progres- sion. Changes in an RNFL defect were determined from serial RNFL photographs and defined as the appearance of a new defect or an increase in the width or depth of an existing defect. These were regarded as indicative of structural progression. Two observers (B.R.S. and J.W.J.), who were blinded to all other patient information, independently evaluated all photographs. In cases of disagreement, a third glaucoma specialist (K.H.P.) served as an adjudicator. Func- tional progression was determined by event analysis using the commercial guided progression analysis (GPA) software provided by the VF device. Functional progression was confirmed when at least three test points were flagged as having significantly deteriorated at the same test point locations in three consecutive fields (the software classifies VF progression as ‘likely pro- gression’) [9, 10]. These changes also had to have been observed at the final visit. 4 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0222166 September 16, 2019 Risk factors associated with recurrent disc hemorrhage PLOS ONE \| https://doi.org/10.1371/journal.pone.0222166 September 16, 2019 Risk factors associated with recurrent disc hemorrhage Table 1. Demographics and baseline data on 178 primary open-angle glaucoma patients. Total (n = 178) Recurrent DH group (n = 89) Control group (n = 89) P-value Age, mean (SD), years 64.18 (12.22) 64.55 (11.49) 63.81 (12.63) 0.683a Male, number (%) 98 (55.06) 47 (52.81) 51 (57.30) 0.547b Follow-up period, mean (SD), months 126.94 (22.16) 119.5 (25.83) 134.43 (14.38) <0.001 a Follow-up number, mean (SD), number 24.7 (4.79) 24.05 (5.28) 23.48 (4.32) 0.486 a Follow-up interval, mean (SD), months 5.60 (1.01) 5.30 (0.89) 5.86 (1.04) 0.001 a CCT, mean (SD), mm 525.04 (39.55) 523.95 (32.52) 526.11 (40.01) 0.738 a AXL, mean (SD), μm 24.24 (1.27) 24.07 (1.07) 24.36 (1.24) 0.242 a Medication number (SD), number 1.40 (0.64) 1.47 (0.66) 1.29 (0.61) 0.060 b Baseline IOP, mean (SD), mmHg 15.59 (3.24) 14.92 (3.63) 16.40 (3.35) 0.010 a Initial VF MD, mean (SD), dB -4.24 (4.59) -4.15 (5.34) -4.33 (4.07) 0.807 a Initial VF PSD, mean (S), dB 5.54 (4.17) 5.35 (4.33) 5.72 (3.87) 0.543 a Average RNFL thickness, mean (SD), μm 88.60 (13.82) 90.31 (13.74) 87.27 (13.47) 0.225a Table 1. Demographics and baseline data on 178 primary open-angle glaucoma patients. SD, standard deviation; CCT, central corneal thickness; AXL, axial length; IOP, intraocular pressure; VF, visual field; MD, mean deviation; PSD, pattern standard deviation; RNFL, retinal nerve fiber layer. a Independent t-test a Independent t-test b Chi-square test; bolded values represent significance, P < 0.05. Results The study included 652 eyes from 652 POAG patients analyzed with color disc photography, red-free RNFL photography, and VF testing. Of these, 60 eyes were excluded because of poor quality red-free RNFL photography or VF testing. In addition, 58 patients who had retinal dis- eases or optic neuropathy and 18 patients who had undergone glaucoma laser treatment or surgery were excluded. Of the remaining 516 POAG patients, 166 showed 1 or 2 occurrences of DH, and 19 patients who had more than 3 occurrences of DH, but the intervals between them was less than 6 months, were also excluded. This left 331 POAG patients. Of these, 89 eyes from 89 POAG patients were included in the recurrent DH group and 89 eyes from 89 POAG patients were included in the age-matched control group. The other 153 patients did not exhibit any DH over the course of follow-up. However, they were not followed-up for more than 10 years because of intermittent visits, or follow-up loss and were excluded from the control group. A total of 178 eyes (89 recurrent DH eyes and 89 control eyes) from 178 POAG patients were included in this study. The patients’ demographic information and base- line characteristics are summarized in Table 1. The mean age was 64.18 ± 12.22 years, and the mean follow-up period was 126.94 ± 22.16 months. Patient age indicates the age at first visit. The recurrent DH group had a shorter follow-up period, more frequent follow-up visits and lower baseline IOP than the no-DH group (P <0.001, 0.001, and 0.010, respectively). Among the ocular factors, baseline IOP (odds ratio [OR], 0.88; 95% confidential interval [CI], 0.80–0.98; P = 0.014) and percentage reduction of IOP (OR, 0.96; 95% CI, 0.93–0.99; P = 0.020) were associated with recurrent DH in the univariate logistic regression analysis (Table 2). Among the systemic factors, cold extremities (OR, 2.80; 95% CI, 1.03–7.60; P = 0.043), a prone or lateral decubitus sleeping position (OR, 2.14; 95% CI, 1.13–4.03; P = 0.019), and sleep disorder (OR, 2.33; 95% CI, 1.05–5.15; P = 0.037) were associated with recurrent DH in the same univariate logistic regression analysis (Table 3). According to both ocular and systemic factors in our multivariate logistic regression analy- sis, percent reduction of IOP (OR, 0.96; 95% CI, 0.93–1.00; P = 0.046) was associated with recurrent DH (Table 4). 5 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0222166 September 16, 2019 g p attern standard deviation; RNFL, retinal nerve fiber layer. DH, disc hemorrhage; OR, odds ratio; CI, confidence interval; SD, standard deviation; CCT, central corneal thickness; AXL, axial length; IOP, intraocular pressure; VF, visual field; MD, mean deviation; PSD, pattern standard deviation; RNFL, retinal nerve fiber layer. nfidence interval; SD, standard deviation; CCT, central corneal thickness; AXL, axial length; IOP, intraocular pressure; VF, SD, standard deviation; CCT, central corneal thickness; AXL, axial length; IOP, intraocular pressure; VF, visual field; MD, mean deviation; PSD, pattern standard deviation; RNFL, retinal nerve fiber layer. DH, disc hemorrhage; OR, odds ratio; CI, confidence interval; SD, standard deviation; CCT, central corneal thickness; AXL, axial length; IOP, intraocular pressure; VF, visual field; MD, mean deviation; PSD, pattern standard deviation; RNFL, retinal nerve fiber layer. lded values represent significance, P < 0.05. DH, disc hemorrhage; OR, odds ratio; CI, confidence interval; SD, standard deviation; CCT, central corneal thickness; AXL visual field; MD, mean deviation; PSD, pattern standard deviation; RNFL, retinal nerve fiber layer. Univariate logistic regression analysis; bolded values represent significance, P < 0.05. eld; MD, mean deviation; PSD, pattern standard deviation; RNFL, retinal nerve fiber layer. DH, disc hemorrhage; OR, odds ratio; CI, confidence interval; SD, standard deviation; CCT, central corneal thickness; AXL SD, standard deviation; CCT, central corneal thickness; AXL, axial length; IOP, intraocular pressure; VF, visual field; MD, m deviation; RNFL, retinal nerve fiber layer. Risk factors associated with recurrent disc hemorrhage Table 3. Univariate logistic regression analysis of systemic factors. Recurrent DH group (n = 89) Control group (n = 89) Univariate analysis OR 95% CI P value Diabetes, No (%) 15 (16.9) 15 (16.9) 1.00 0.46–2.19 1.000 Systemic hypertension, No (%) 36 (40.4) 26 (29.2) 1.65 0.88–3.07 0.117 Cardiac disease, No (%) 11 (12.4) 13 (14.6) 0.82 0.35–1.95 0.661 Aspirin or anticoagulant, No (%) 17 (19.1) 18 (20.2) 1.07 0.51–2.25 0.850 Steroid use, No (%) 1 (1.1) 1 (1.1) 1.00 0.06–16.24 1.000 Gingko extract, No (%) 3 (3.4) 2 (2.2) 1.52 0.25–9.31 0.652 Ocular operation history, No (%) 20 (22.5) 21 (23.6) 0.94 0.47–1.89 0.859 Ocular trauma history, No (%) 0 1 (1.1) 1.00 0 1.000 Cold extremities, No (%) 15 (16.9) 6 (6.7) 2.80 1.03–7.60 0.043 Migraine, No (%) 7 (7.9) 5 (5.6) 1.43 0.44–4.70 0.552 Glaucoma family history, No (%) 8 (9.0) 8 (9.0) 1.00 0.36–2.79 1.000 Sleeping position (prone/ lateral decubitus), No (%) 38 (42.7) 23 (25.8) 2.14 1.13–4.03 0.019 Yoga, No (%) 5 (5.6) 4 (4.5) 1.27 0.33–4.87 0.733 Sleep disorder (sleep apnea/snoring), No (%) 22 (24.7) 11 (12.4) 2.33 1.05–5.15 0.037 Constipation, No (%) 2 (2.2) 1 (1.1) 2.02 0.18–22.72 0.568 Heavy load, No (%) 4 (4.5) 6 (6.7) 0.65 0.18–2.39 0.518 Wind instrument playing, No (%) 1 (1.1) 1 (1.1) 1.00 0.06–16.24 1.000 DH, disc hemorrhage; OR, odds ratio; CI, confidence interval. Univariate logistic regression analysis; bolded values represent significance, P < 0.05. https://doi org/10 1371/journal pone 0222166 t003 Table 3. Univariate logistic regression analysis of systemic factors. Glaucoma progression p g The Kaplan-Meier survival analysis revealed that patients in the control group had a higher probability for cumulative non-progression than patients in the recurrent DH group (P = 0.01, log-rank test) (Fig 1). The multivariate Cox proportional-hazards regression models revealed that recurrent DH was significantly associated with glaucoma progression (hazard ration (HR), 1.88, 95% CI, 1.16–3.05; P = 0.01) (Table 5). The Kaplan-Meier survival analysis revealed that patients in the control group had a higher probability for cumulative non-progression than patients in the recurrent DH group (P = 0.01, log-rank test) (Fig 1). The multivariate Cox proportional-hazards regression models revealed that recurrent DH was significantly associated with glaucoma progression (hazard ration (HR), 1.88, 95% CI, 1.16–3.05; P = 0.01) (Table 5). Table 2. Univariate logistic regression analysis of ocular factors. Table 2. Univariate logistic regression analysis of ocular factors. Recurrent DH group (n = 89) Control group (n = 89) Univariate analysis OR 95% CI P value CCT, mean (SD), mm 523.95 (32.52) 526.11 (40.01) 1.00 0.99–1.01 0.736 AXL, mean (SD), μm 24.07 (1.07) 24.36 (1.24) 0.80 0.55–1.16 0.241 Baseline IOP, mean (SD), mmHg 14.92 (3.63) 16.40 (3.35) 0.88 0.80–0.98 0.014 IOP with medication, mean (SD), mmHg Mean IOP, mean (SD), mmHg 13.51 (2.50) 13.56 (2.22) 0.99 0.85–1.16 0.929 Fluctuation of IOP, mean (SD), mmHg 1.96 (1.50) 1.87 (0.67) 1.10 0.78–1.54 0.588 Maximum IOP, mean (SD), mmHg 16.99 (5.78) 18.03 (4.06) 0.95 0.87–1.04 0.244 Percentage reduction of IOP, mean (SD), % 11.50 (12.81) 17.00 (10.87) 0.96 0.93–0.99 0.020 Initial VF MD, mean (SD), dB -4.15 (5.34) -4.33 (4.07) 1.01 0.95–1.07 0.806 Initial VF PSD, mean (SD), dB 5.35 (4.33) 5.72 (3.87) 0.98 0.91–1.05 0.541 Average RNFL thickness, mean (SD), μm 90.31 (13.74) 87.27 (13.47) 1.02 0.99–1.05 0.226 PLOS ONE \| https://doi.org/10.1371/journal.pone.0222166 September 16, 2019 6 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0222166 September 16, 2019 Discussion The results of our evaluation of the risk factors for recurrent DH in POAG patients demon- strated an association with lower percent reduction of IOP. The strength of our study is its nar- row definition of recurrent DH. In the recurrent DH group, we included only eyes showing an occurrence of DH of at least 3 times (recurrent DH) during the follow-up period, and in the control group, only eyes that did not show DH over the course of at least 10 years of follow-up were included. Note that previous studies compared recurrent-DH (with an occurrence of DH of at least 2 times) eyes and no-DH eyes or between recurrent-DH eyes and single-DH eyes [7, 10–12]. Our more strict criteria for the DH and control groups were formulated for the pur- poses of more clearly isolating the risk factors for the occurrence of DH. Table 4. Multivariate logistic regression analysis of ocular and systemic factors. Model 1 Model 2 OR 95% CI P value OR 95% CI P value Baseline IOP mmHg 1.01 0.85–1.21 0.899 Percent reduction of IOP % 0.97 0.92–1.01 0.181 0.96 0.93–1.00 0.046 Cold extremities No 3.21 0.73–14.18 0.123 3.52 0.82–15.11 0.090 Sleeping position (prone/lateral decubitus) No 1.96 0.80–4.83 0.142 Sleep disorder (sleep apnea/ snoring) No 1.83 0.61–1.65 0.279 Table 4. Multivariate logistic regression analysis of ocular and systemic factors. Table 4. Multivariate logistic regression analysis of ocular and systemic factors. Discussion Model 1 Model 2 OR 95% CI P value OR 95% CI P value Baseline IOP mmHg 1.01 0.85–1.21 0.899 Percent reduction of IOP % 0.97 0.92–1.01 0.181 0.96 0.93–1.00 0.046 Cold extremities No 3.21 0.73–14.18 0.123 3.52 0.82–15.11 0.090 Sleeping position (prone/lateral decubitus) No 1.96 0.80–4.83 0.142 Sleep disorder (sleep apnea/ snoring) No 1.83 0.61–1.65 0.279 OR, odds ratio; CI, confidence interval.; IOP, IOP, intraocular pressure Multivariate logistic regression analysis; bolded values represent significance, P < 0.05 Model 1 includes all of the risk factors with p < 0.10 in univariate analysis Model 2 includes risk factors selected by backward stepwise method started with items in model 1 https://doi.org/10.1371/journal.pone.0222166.t004 Baseline IOP mmHg 1.01 0.85–1.21 0.899 Percent reduction of IOP % 0.97 0.92–1.01 0.181 0.96 0.93–1.00 0.046 Cold extremities No 3.21 0.73–14.18 0.123 3.52 0.82–15.11 0.090 Sleeping position (prone/lateral decubitus) No 1.96 0.80–4.83 0.142 Sleep disorder (sleep apnea/ snoring) No 1.83 0.61–1.65 0.279 OR, odds ratio; CI, confidence interval.; IOP, IOP, intraocular pressure Multivariate logistic regression analysis; bolded values represent significance, P < 0.05 Model 1 includes all of the risk factors with p < 0.10 in univariate analysis Model 2 includes risk factors selected by backward stepwise method started with items in model 1 h //d i /10 1371/j l 0222166 004 OR, odds ratio; CI, confidence interval.; IOP, IOP, intraocular pressure PLOS ONE \| https://doi.org/10.1371/journal.pone.0222166 September 16, 2019 7 / 12 7 / 12 Risk factors associated with recurrent disc hemorrhage Fig 1. Kaplan-Meier curves comparing cumulative non-progression probability between the recurrent DH and control groups. The statistical endpoint was defined as the time of the first indication of structural or functional glaucoma progression. The control group had a greater cumulative non-progression probability than the recurrent DH group (P = 0.01, log-rank test). https://doi.org/10.1371/journal.pone.0222166.g001 Fig 1. Kaplan-Meier curves comparing cumulative non-progression probability between the recurrent DH and control groups. The statistical endpoint was defined as the time of the first indication of structural or functional glaucoma progression. The control group had a greater cumulative non-progression probability than the recurrent DH group (P = 0.01, log-rank test). https://doi.org/10.1371/journal.pone.0222166.g001 https://doi.org/10.1371/journal.pone.0222166.g001 The relationship between IOP and DH remains controversial [6, 13–15]. Soares et al. [16] reported IOP at first DH detection to be lower than the mean IOP of the three most recent vis- its; and, Jonas et al. HR, hazard ratio; CI, confidence interval; IOP, intraocular pressure; VF, visual field; MD, mean deviation; PSD, pattern standard deviation; DH, disc hemorrhage Model 1 includes all of the risk factors with p <0.10 in univariate analysis he risk factors with p <0.10 in univariate analysis ; IOP, intraocular pressure; VF, visual field; MD, mean deviation; PSD, pattern standard deviation; DH, disc hemorrhage y ward stepwise method started with items in model 1 p y tors selected by backward stepwise method started with items in model 1 azards regression model; bolded values represent significance, P < 0.05 Model 2 includes the risk factors selected by backward stepwise method started with items in model 1 ; p g , 10 in univariate analysis Discussion This might be due to the relationship between sleeping position and IOP. Recently, several studies have reported that in the lateral decubitus position, IOP was higher in the dependent eye (lower eye) than in the non-dependent eye (upper eye) [19–21]. Further- more, among the various sleeping positions, IOP is known to be highest in the prone position. Previously, we reported that no preferred sleeping position (i.e., neither of the one-sided posi- tions) was specifically associated with unilateral DH in normal-tension glaucoma (NTG) [22]. A number of previous studies have evaluated variable associations with DH in glaucoma patients and have suggested several risk factors [5, 16, 23]. However, no study investigating the risk factors for recurrent DH has been conducted. The most frequently cited risk factors are diabetes, systemic hypertension, increasing IOP, and the use of aspirin. However, in our study, diabetes, systemic hypertension, and the use of aspirin were found to be unrelated to recurrent DH. None showed a significant relationship with recurrent DH, even in the univariate logistic regression analysis. With regard to the effect of aspirin on DH, a number of studies have inves- tigated this relationship. Recently, Shim et al. [24] found a significant association between platelet function and DH in glaucoma patients. According to them, that aspirin prevents thromboxane A2 production, which is known to promote platelet aggregation, and this mech- anism, through the use of aspirin, can both increase the risk of DH and delay its absorption. However, use of aspirin was not a related factor in our study. Also, it should be noted that the effect of aspirin on platelet function can vary according to dosage levels. IOP reduction and DH recurrence may be related, but it is difficult to assume a causal rela- tionship. However, in this study, we performed a logistic regression analysis with various ocu- lar and systemic factors to determine which ones might be associated with recurrent DH. According to the results, DH was a relevant factor. We attempted to control the IOP of the patients both with and without DH. Nevertheless, we found that recurrent DH was more com- mon in patients with relatively low IOP reductions. This suggests that IOP might be a risk fac- tor for recurrent DH. In addition, this study demonstrated that recurrent DH was also associated with the progression of glaucoma. Discussion [17] suggested that eyes with high IOP had the possibility of earlier bleed- ing stops. However, Miyake et al. [18] noted that glaucoma patients showed a decreased incidence of DH after trabeculectomy. To our knowledge, ours is the first study to evaluate the association between IOP-related factors such as percent reduction of IOP and recurrent DH. In our results, a lower percent reduction of IOP was the risk factor for recurrent DH. From this, we deduced that IOP reduction is essential for DH-rate reduction. However, we still do not understand the underlying mechanism behind the association between recurrent DH and IOP reduction. One possibility is ischemic microinfarction in the optic disc or the mechanical Table 5. Univariate and multivariate Cox proportional hazards regression models’ data for the prediction of progression. Univariate Multiple Model 1 Model 2 HR 95% CI P value HR 95% CI P value HR 95% CI P value Baseline IOP 0.99 0.91–1.07 0.72 Percentage reduction of IOP 0.97 0.95–1.00 0.06 0.98 0.95–1.01 0.12 Initial VF MD 0.98 0.94–1.03 0.41 Initial VF PSD 1.05 0.99–1.10 0.08 1.08 1.02–1.15 0.01 1.05 1.00–1.10 0.06 Recurrent DH 1.87 1.15–3.02 0.01 2.27 1.22–4.23 0.01 1.88 1.16–3.05 0.01 Age 1.00 0.98–1.02 0.73 Sex 1.30 0.81–2.08 0.27 Diabetes 1.29 0.68–2.26 0.49 Hypertension 0.74 0.44–1.24 0.26 Cold extremities 1.69 0.90–3.14 0.10 Sleeping position (prone/ lateral decubitus) 0.88 0.52–1.47 0.62 Sleep disorder (sleep apnea/snoring) 0.95 0.52–1.74 0.88 ortional hazards regression models’ data for the prediction of progression. le 5. Univariate and multivariate Cox proportional hazards regression models’ data for the prediction of progression. PLOS ONE \| https://doi.org/10.1371/journal.pone.0222166 September 16, 2019 8 / 12 Risk factors associated with recurrent disc hemorrhage rupture of small blood vessels caused by IOP-change-related structural alterations in the lam- ina cribrosa. Nonetheless, further study is required to uncover the pathophysiology of DH and the mechanism behind the correlation between IOP reduction and recurrent DH. As indicated in Table 5, a prone or lateral decubitus sleeping position was not an associated factor in the multivariate analysis. However, it was an associated factor in the univariate analy- sis (Table 3). This might be due to the relationship between sleeping position and IOP. As indicated in Table 5, a prone or lateral decubitus sleeping position was not an associated factor in the multivariate analysis. However, it was an associated factor in the univariate analy- sis (Table 3). PLOS ONE \| https://doi.org/10.1371/journal.pone.0222166 September 16, 2019 Discussion This confirms that the IOP reduction is clinically significant for the prevention of glaucoma progression. IOP reduction and DH recurrence may be related, but it is difficult to assume a causal rela- tionship. However, in this study, we performed a logistic regression analysis with various ocu- lar and systemic factors to determine which ones might be associated with recurrent DH. Our study has a number of limitations. First, we might have missed some DH cases that had ‘developed and absorbed’ between visits. However, to overcome this limitation, we included, as the control group, only patients who had at least 10 years of follow-up. Moreover, we previously reported that most cases of DH were detected within 5 years of follow-up and that there was no detection-rate increase thereafter [25]. Therefore, the likelihood that we missed any DH incidences is quite low. In addition, recurrent DH was defined as an interval between DH onset and a period of at least 6 months. By doing so, we prevented the erroneous recording of cases of DH non-absorbance as additional DH occurrences. Second, the recurrent DH group exhibited a longer follow-up time and more frequent follow-ups than the no-DH group. One of the reasons for the shorter follow-up interval for the DH group was that those patients were frequently followed-up after the detection of DH. However, once it had been confirmed that DH was absorbed, the follow-up was not significantly different from no-DH cases. Furthermore, the follow-up period was shorter in the recurrent DH group than the con- trol group. However, a mean of 126 months is considered to be a sufficient period of time for the effect of glaucoma medication to manifest. Third, some factors, such as diabetes control, can change over time. The difference in the follow-up period between the two groups might, PLOS ONE \| https://doi.org/10.1371/journal.pone.0222166 September 16, 2019 9 / 12 Risk factors associated with recurrent disc hemorrhage therefore, be a factor affecting the evaluation of some factors. To minimize this limitation, we excluded patients who had systemic factor changes referenced in their medical records and in the telephone surveys. Fourth, there is theoretical and clinical support for a relationship between baseline IOP and the ability to reduce IOP with treatment [26–28], and it can be diffi- cult to separate the effect of these two variables. Supporting information S1 Table. Glaucoma progression. (XLSX) S1 Table. Glaucoma progression. (XLSX) Acknowledgments We would like to thank Editage (www.editage.co.kr) for English language editing. Author Contributions Conceptualization: Ki Ho Park. Data curation: Bo Ram Seol, Jin Wook Jeoung, Ki Ho Park. Data curation: Bo Ram Seol, Jin Wook Jeoung, Ki Ho Park. Supervision: Jin Wook Jeoung, Ki Ho Park. Writing – original draft: Bo Ram Seol. Writing – original draft: Bo Ram Seol. Discussion Thus, in this study, the multicollinearity between baseline IOP and percent reduction of IOP was assessed by calculating the VIFs. In the results, the VIF value was 1.001 for both baseline IOP and the percent reduction of IOP. This finding suggests that there is no multicollinearity between baseline IOP and percent reduction of IOP. Fifth, the telephone survey itself has limitations. This tool depends on respondents’ memories and possibly subjective answers, and, as such, there is always a possi- bility that they might not completely or clearly understand their systemic conditions. Addi- tionally, the possibility of such a mild disease being unrecognized by patients is a limitation of most studies conducted via telephone survey. Sixth, trauma history and steroid use were ana- lyzed with only a few data points, so the predictive power may be weak. Seventh, some of the systemic factors used in this study, such as constipation, carrying heavy loads, and wind- instrument playing, can have ambiguous definitions. In conclusion, our study demonstrates that POAG patients with a lower percent reduction of IOP were at higher risk for the development of recurrent DH. Among ocular and systemic factors, only lower percent reduction of IOP was associated with recurrent DH. This finding suggests that less IOP reduction from baseline might be associated with the recurrence of DH in POAG. PLOS ONE \| https://doi.org/10.1371/journal.pone.0222166 September 16, 2019 References 1. Drance S, Anderson DR, Schulzer M, Group CN-TGS. Risk factors for progression of visual field abnor- malities in normal-tension glaucoma. Am J Ophthalmol. 2001; 131(6):699–708. https://doi.org/10.1016/ s0002-9394(01)00964-3 PMID: 11384564 1. 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Clinical factors associated with pro- gression of glaucomatous optic disc damage in treated patients. Arch Ophthalmol. 2001; 119(6):813–8. https://doi.org/10.1001/archopht.119.6.813 PMID: 11405831 16. Soares AS, Artes PH, Andreou P, Leblanc RP, Chauhan BC, Nicolela MT. Factors associated with optic disc hemorrhages in glaucoma. Ophthalmology. 2004; 111(9):1653–7. https://doi.org/10.1016/j.ophtha. 2004.03.023 PMID: 15350318 17. Jonas JB, Xu L. Optic disk hemorrhages in glaucoma. Am J Ophthalmol. 1994; 118(1):1–8. https://doi. org/10.1016/s0002-9394(14)72835-1 PMID: 8023857 18. Miyake T, Sawada A, Yamamoto T, Miyake K, Sugiyama K, Kitazawa Y. Incidence of disc hemorrhages in open-angle glaucoma before and after trabeculectomy. J Glaucoma. 2006; 15(2):164–71. PMID: 16633231 19. Malihi M, Sit AJ. Effect of head and body position on intraocular pressure. Ophthalmology. 2012; 119 (5):987–91. https://doi.org/10.1016/j.ophtha.2011.11.024 PMID: 22341914 20. Lee JY, Yoo C, Jung JH, Hwang YH, Kim YY. The effect of lateral decubitus position on intraocular pres- sure in healthy young subjects. Acta Ophthalmol. 2012; 90(1):e68–e72. https://doi.org/10.1111/j.1755- 3768.2011.02208.x PMID: 21801338 21. Kim KN, Jeoung JW, Park KH, Lee DS, Kim DM. Effect of lateral decubitus position on intraocular pres- sure in glaucoma patients with asymmetric visual field loss. Ophthalmology. 2013; 120(4):731–5. https://doi.org/10.1016/j.ophtha.2012.09.021 PMID: 23260257 22. Kim KN, Park KH. Relationship between preferred sleeping position and unilateral disc haemorrhage in normal-tension glaucoma patients. Ophthalmology. 2015; 120:731–5. 23. Kim Y, Han S, Park K, Kim S, Kim S, Seong M, et al. Risk factors associated with optic disc haemor- rhage in patients with normal tension glaucoma. Eye. 2010; 24(4):567–72. https://doi.org/10.1038/eye. 2009.163 PMID: 19648906 11 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0222166 September 16, 2019 Risk factors associated with recurrent disc hemorrhage 24. Shim SH, Kim JM, Woo H-Y, Shin KU, Koh JW, Park KH. Association between platelet function and disc hemorrhage in patients with normal-tension glaucoma: a prospective cross-sectional study. Am J Ophthalmol. 2015; 160(6):1191–9. e1. https://doi.org/10.1016/j.ajo.2015.09.006 PMID: 26384167 25. Suh MH, Park KH. Period prevalence and incidence of optic disc haemorrhage in normal tension glau- coma and primary open-angle glaucoma. Clin Exp Ophthalmol. 2011; 39(6):513–9. https://doi.org/10. 1111/j.1442-9071.2010.02482.x PMID: 21176041 26. Musch DC, Gillespie BW, Niziol LM, Cashwell LF, Lichter PR. Factors associated with intraocular pres- sure before and during 9 years of treatment in the Collaborative Initial Glaucoma Treatment Study. Oph- thalmology. 2008; 115(6):927–33. https://doi.org/10.1016/j.ophtha.2007.08.010 PMID: 17964655 27. Heijl A, Leske MC, Hyman L, Yang Z, Bengtsson B. Intraocular pressure reduction with a fixed treat- ment protocol in the Early Manifest Glaucoma Trial. Acta Ophthalmol. 2011; 89(8):749–54. https://doi. org/10.1111/j.1755-3768.2009.01852.x PMID: 20236252 28. Ang GS, Kersey JP, Shepstone L, Broadway DC. The effect of travoprost on daytime intraocular pres- sure in normal tension glaucoma: a randomised controlled trial. Br J Ophthalmol. 2008; 92(8):1129–33. https://doi.org/10.1136/bjo.2007.135269 PMID: 18511540 12 / 12
https://openalex.org/W4292470359	https://minerva.autanabooks.com/index.php/Minerva/article/download/63/209	Spanish; Castilian	null	Multivariate analysis of the impact and interdependence of teleworking with variables of productivity, efficiency, effectiveness, job satisfaction and knowledge in digital tools: a case study.	Minerva (Quito)	2,022	cc-by	4,368	Minerva Journal Vol.3, Issue. 8, (pp. 42-53) Minerva Journal Vol.3, Issue. 8, (pp. 42-53) Minerva Journal Vol.3, Issue. 8, (pp. 42-53) ISSN-E: 2697-3650 https://doi.org/10.47460/minerva.v3i8.63 Análisis multivariante del impacto e interdependencia del teletrabajo con variables de productividad, eficiencia, eficacia, satisfacción laboral y conocimiento en herramientas digitales: un caso de estudio. https://doi.org/10.47460/mine Alviarez Jesica direccionejecutiva.coachorg@gmail.com https://orcid.org/0000-0003-3124-2961 Unexpo Puerto Ordaz Estado Bolívar, Venezuela I. INTRODUCCIÓN La crisis sanitaria mundial generada por la pandemia del COVID-19 ha logrado impactar considerablemente las estructuras organizacionales, desde las transformaciones radicales de sus naturalezas hasta sus organizaciones y sistemas, por lo que las mismas han buscado la forma de adaptarse a los vertiginosos cambios dinámicos y demandantes de los consumidores. Desde las Tecnologías de la Información y la Comunicación (TICs) las organizaciones han evolucionado a nuevos modelos productivos, lo que ha implicado a su vez cambios importantes en sus estructuras internas [1]. Así, las organizaciones tradicionales han adaptado de forma brusca sus metodologías de trabajo, la medición de objetivos, los resultados y entregables a la nueva realidad de teletrabajo o trabajo remoto; en la cual el desempeño se ha visto significativamente impactado. Por ello, se desea determinar los componentes principales de las variables observables organizacionales relacionadas con el teletrabajo a partir de un caso de estudio vinculado a una empresa automotriz japonesa; para lo cual se hará uso del software IBM SPSS a fines de llevar a cabo el análisis multivariante y la simulación de la data referencial sobre los indicadores de productividad, eficiencia, eficacia, satisfacción laboral y, de esta manera, obtener de la regresión lineal los coeficientes de los factores con mayores pesos de las varianzas asociadas a la variable del teletrabajo [2]. El presente caso de estudio contiene como partes del trabajo: el desarrollo, la metodología, los resultados y conclusiones, así como las referencias bibliográficas de soporte. Recibido(12/04/2022), Aceptado(30/04/2022) Resumen—A partir de la revisión e investigación sobre la modalidad del teletrabajo y las variables que lo impactan a nivel organizacional, como calidad de vida, comunicación, cultura organizacional o productividad, se propone un caso de estudio bajo el modelo de innovación y teletrabajo de una empresa automotriz japones para la aplicación de técnicas de Análisis Multivariantes como el Análisis de Componentes Principales y la Regresión lineal, a fines de condensar la información aportada por múltiples variables en componentes principales y validar las relaciones e impacto que existen entre ellas, determinando así la interdependencia y correlación de las mismas con la variable teletrabajo, permitiendo simplificar la complejidad de espacios muestrales con muchas dimensiones. Se lograron identificar los componentes principales con etiquetas propias de las variables y se validaron estadísticamente las variables dependientes y predictivas del caso, a través del uso del software IBM SPSS. Palabras clave: teletrabajo, análisis multivariante, componentes principales. Multivariate analysis of the impact and interdependence of teleworking with variables of productivity, efficiency, effectiveness, job satisfaction and knowledge in digital tools: a case study. Abstract—- Based on the review and research on the teleworking modality and the variables that impact it at the organizational level, such as quality of life, communication, organizational culture or productivity, a case study is proposed under the innovation and teleworking model of a company. Japanese automotive industry for the application of Multivariate Analysis techniques such as Principal Component Analysis and Linear Regression, in order to condense the information provided by multiple variables into principal components and validate the relationships and impact that exist between them, thus determining the interdependence and correlation of the same with the telework variable, allowing to simplify the complexity of sample spaces with many dimensions. It was possible to identify the main components with the variables' own labels and the dependent and predictive variables of the case were statistically validated, through the use of the IBM SPSS software. Keywords: telecommuting, multivariate analysis, principal components. Alviarez. Análisis multivariante del impacto e interdependencia del teletrabajo con variables de productividad, eficiencia, eficacia, satisfacción laboral y conocimiento en herramientas digitales: un caso de estudio. 42 42 Minerva Journal Vol.3, Issue. 8, (pp. 42-53) ISSN-E: 2697-3650 Fig. 2 Modelo propuesto para el Análisis Multivariable Es importante destacar, que las variables latentes o constructos (componentes principales) son aquellos que no son directamente observables o medibles, por lo que no presentan una unidad de medida específica, y en consecuencia, cualquier estimación supone un error asociado. Mientras que las variables observables son un conjunto de medidas que se utilizan para definir o inferir la variable latente [5]. II. DESARROLLO El teletrabajo consiste en una modalidad laboral desarrollada a distancia (fuera de la organización) a través de las tecnologías de la información y las comunicaciones (TIC), lo que genera el desplazamiento del “producto” hasta el sitio de interés en vez del individuo. Si la producción y los entregables del empleado son de carácter intelectual, la realización de las asignaciones y las tareas laborales podrían efectuarse desde cualquier locación (siempre y cuando se tenga acceso a las TIC) [3]. En el proceso de investigación sobre el tema de teletrabajo, se consigue visualizar el uso de variedades de términos que hacen alusión a la definición de la modalidad del trabajo remoto, como los vocablos telework, telecommuting (teledesplazamiento), networking (trabajo en red), remote working (trabajo a distancia), flexible working (trabajo flexible), o home working (trabajo a domicilio) [4]. Existen diversos estudios y autores que proponen el vínculo entre algunas variables observables entorno al ámbito laboral – bienestar (personal) con el teletrabajo. Entre ellos, [3] plantea el teletrabajo con la calidad de vida y la productividad como resultado de una relación recursiva así como un modelo organizacional de teletrabajo que comprenden dichos elementos esenciales objeto de estudio. Otra investigación analizada incorpora un elemento adicional vinculado a las TIC´s y a las comunicaciones, en la cual se plantea el impacto del teletrabajo en la cultura organizacional y las posibilidades de intervención desde el campo de la tecnología con la evolución de la modalidad de teletrabajo [1]. De igual forma, se consigue un modelo de ecuaciones estructurales puesto en prueba a través de un modelo predictivo, en donde se detectan que los factores demandantes, recursos laborales y recursos personales que inciden significativamente en la productividad percibida por el empleado (afectada por el estrés) y su satisfacción laboral [5]. El proceso técnico generado para el presente caso de estudio de teletrabajo consiste en un modelo de estructura organizacional basado en la propuesta de tres (03) variables observables: la transformación del negocio, la evolución de la cultura organizacional y la innovación como estilo de trabajo, el cual puede visualizarse a continuación: 4 Alviarez. Análisis multivariante del impacto e interdependencia del teletrabajo con variables de productividad, eficiencia, eficacia, satisfacción laboral y conocimiento en herramientas digitales: un caso de estudio. 43 Minerva Journal Vol.3, Issue. 8, (pp. 42-53) ISSN-E: 2697-3650 Fig. 1 Modelo de nuevo estilo organizacional bajo el enfoque del teletrabajo Fig. II. DESARROLLO 1 Modelo de nuevo estilo organizacional bajo el enfoque del teletrabajo Para ello, se identificaron las variables observadas vinculadas de forma interdependiente con los siguientes indicadores: eficiencia, eficacia, productividad y satisfacción laboral, para lo cual se propone el siguiente esquema de categorización de variables: Fig. 2 Modelo propuesto para el Análisis Multivariable III. METODOLOGÍA La investigación se llevó a cabo con la aplicación de las técnicas multivariadas que permitieron una aproximación de los datos dentro de un marco interpretativo, captar las complejidades e interrelaciones del fenómeno para así proveer un marco teórico que vislumbren los componentes principales [5]. Por ello, para el desarrollo del estudio de caso se aplicó el análisis de datos interdependientes denominada Análisis de Componentes Principales (ACP), la cual permite abordar de forma simultánea múltiples medidas de los objetos bajo observación, creación de conocimiento y el mejoramiento en la toma de decisiones dentro de las organizaciones [6]. 44 Alviarez. Análisis multivariante del impacto e interdependencia del teletrabajo con variables de productividad, eficiencia, eficacia, satisfacción laboral y conocimiento en herramientas digitales: un caso de estudio. 44 Minerva Journal Vol.3, Issue. 8, (pp. 42-53) Minerva Journal Vol.3, Issue. 8, (pp. 42-53) ISSN-E: 2697-3650 De esta forma, el ACP busca hallar combinaciones lineales de las variables originales que expliquen la mayor parte de la variación total. El primer factor o componente será aquel que determina la mayor parte de la varianza total y el segundo factor la mayor parte de la varianza restante, sucesivamente la relación de peso entre varianzas [7]. También, ACP es considerado un análisis de tipo descriptivo con la función primordial es reducir el número de variables existentes, excluyendo aquellas que tengan menos influencia a la hora de establecer diferencias entre los individuos [8]. Para estudiar las relaciones que se presentan entre p variables correlacionadas (que miden información común) se puede transformar el conjunto original de variables en otro conjunto de nuevas variables incorrelacionadas entre sí (que no tenga repetición o redundancia en la información) llamado conjunto de componentes principales [9]. A. Cálculo de los componentes principales: Se considera una serie de variables (x1, x2, ... , xp) sobre un grupo de objetos o individuos y se trata de calcular, a partir de ellas, un nuevo conjunto de variables (y1, y2, ... , yp), incorrelacionadas entre sí, cuyas varianzas vayan decreciendo progresivamente, generándose la siguiente ecuación matemática [9]: Siendo un vector de constantes y se presenta como Para este caso, la metodología a seguir radica en que cada componente principal será expresado como función de p variables observadas correlacionadas entre sí, que se ponderan en dicho componente, mediante la siguiente expresión matemática [7]: Para este caso, la metodología a seguir radica en que cada componente principal será expresado como función de p variables observadas correlacionadas entre sí, que se ponderan en dicho componente, mediante la siguiente expresión matemática [7]: Donde: = pesos o coeficientes de saturación X= variables, si están estandarizadas se sustituyen por Z [7]. De forma gráfica, el ACP se visualiza de la siguiente manera: Fig. 3 Matriz Variables Latentes vs Variables Observadas 45 Donde: = pesos o coeficientes de saturación X= variables, si están estandarizadas se sustituyen por Z [7]. De forma gráfica, el ACP se visualiza de la siguiente manera: Fig. 3 Matriz Variables Latentes vs Variables Observadas Alviarez. Análisis multivariante del impacto e interdependencia del teletrabajo con variables de productividad, eficiencia, eficacia, satisfacción laboral y conocimiento en herramientas digitales: un caso de estudio. = pesos o coeficientes de saturación X= variables, si están estandarizadas se sustituyen por Z [7]. De forma gráfica, el ACP se visualiza de la siguiente manera: Fig. 3 Matriz Variables Latentes vs Variables Observadas 45 Alviarez. Análisis multivariante del impacto e interdependencia del teletrabajo con variables de productividad, eficiencia, eficacia, satisfacción laboral y conocimiento en herramientas digitales: un caso de estudio. 4 Alviarez. Análisis multivariante del impacto e interdependencia del teletrabajo con variables de productividad, eficiencia, eficacia, satisfacción laboral y conocimiento en herramientas digitales: un caso de estudio. 45 Minerva Journal Vol.3, Issue. 8, (pp. Primero, se buscaron los estadísticos descriptivos de la data en estudio: rimero, se buscaron los estadísticos descriptivos de la data en estudio: Los descriptores univariados incluyen la media, la desviación estándar y el número de casos válidos para cada variable. La solución inicial muestra las comunalidades iniciales, los autovalores y el porcentaje de varianza explicada [10]. Matriz de correlaciones. Para el caso de estudio se aplicarán KMO y prueba de esfericidad de Bartlett., y la reproducida a fines de contrastar los resultados obtenidos. B. Reducción de Dimensiones (Factor) -ACP. Se procede a la reducción de dimensiones a través de la función factor, estableciéndose como método de análisis el ACP para el proceso de extracción. De igual forma, se configura el análisis para la generación de una matriz de correlación, inicialmente sin la rotación del factor de solución y se especifica un número máximo de correlaciones por convergencia de 25. De esta forma, se seleccionan las variables observadas objeto del presente estudio, obteniéndose así las comunalidades y las varianzas para su respectivo análisis, así como la matriz de Componentes principales, la cual nos presenta la solución factorial y estará constituida por las correlaciones entre las variables originales o saturaciones para cada componente o factor: A. Cálculo de los componentes principales: 42-53) ISSN-E: 2697-3650 Para el análisis de los componentes principales del caso se hizo uso del software IBM SPSS, para la corrida de la muestra de datos y variables observadas como: antigüedad del empleado, resultado de evaluación de desempeño, nivel del cargo, condición laboral (fijo – contratado), tipo de clase de nómina (gerencial, operativa), edad, sexo, departamento laboral, Horas anuales de formación técnica, asistencia promedio al mes y días de ausentismo al mes, el % de conocimientos de herramientas digitales,% de productividad (horas laboradas vs entregables generados), % de eficiencia con base a objetivos alcanzados, % de eficacia (optimización de recursos) y el % de satisfacción laboral. C. Análisis Factorial. El análisis factorial busca identificar variables subyacentes, o factores, que expliquen la configuración de las correlaciones dentro de un conjunto de variables observadas. El análisis factorial se suele utilizar en la reducción de los datos para identificar un pequeño número de factores que explique la mayoría de la varianza observada en un número mayor de variables manifiestas. Dicho procedimiento de análisis factorial ofrece un alto grado de flexibilidad y métodos de rotación [10]. D.Tipo de datos a analizar: ·Datos. Las variables son cuantitativas a nivel de intervalo o de razón. ·Supuestos. Los datos tienen una distribución normal bivariado para cada pareja de variables y las observaciones son independientes. El modelo de análisis factorial especifica que las variables vienen determinadas por los factores comunes (los factores estimados por el modelo) y por factores exclusivos (los cuales no se superponen entre las distintas variables observadas); las estimaciones calculadas se basan en el supuesto de que ningún factor único está correlacionado con los demás, ni con los factores comunes [10]. 46 Alviarez. Análisis multivariante del impacto e interdependencia del teletrabajo con variables de productividad, eficiencia, eficacia, satisfacción laboral y conocimiento en herramientas digitales: un caso de estudio. 46 Minerva Journal Vol.3, Issue. 8, (pp. 42-53) ISSN-E: 2697-3650 IV. RESULTADOS A través del software SPSS se obtuvieron los siguientes resultados, a partir del Análisis de Componentes Principales: A través del software SPSS se obtuvieron los siguientes resultados, a partir del Análisis de Componentes Principales: A. Descriptores univariados: Se obtuvieron los estadísticos descriptivos de la tabla 1: Tabla 1. Estadísticos Descriptivos A continuación, se presenta la matriz de correlación: Tabla 2. Matriz de correlación Se obtuvieron los estadísticos descriptivos de la tabla 1: Tabla 1. Estadísticos Descriptivos Tabla 1. Estadísticos Descriptivos A continuación, se presenta la matriz de correlación: Tabla 2. Matriz de correlación Tabla 2. Matriz de correlación Alviarez. Análisis multivariante del impacto e interdependencia del teletrabajo con variables de productividad, eficiencia, eficacia, satisfacción laboral y conocimiento en herramientas digitales: un caso de estudio. 47 Alviarez. Análisis multivariante del impacto e interdependencia del teletrabajo con variables de productividad, eficiencia, eficacia, satisfacción laboral y conocimiento en herramientas digitales: un caso de estudio. 47 Minerva Journal Vol.3, Issue. 8, (pp. 42-53) ISSN-E: 2697-3650 En la tabla anterior, se logra visualizar el valor del determinante en 0.268. Los determinantes próximos a cero, como resultante del caso en estudio, está indicando que las variables utilizadas están linealmente relacionadas, lo que significa que el análisis factorial, es una técnica pertinente para analizar esas variables B.Comunalidades: La Comunalidad asociada a la variable j‐ésima es la proporción de variabilidad de dicha variable explicada por los k factores considerados. Se pueden visualizar las comunalidades asignadas a las variables (inicial) y la extracción vinculada a las variables generadas por la solución factorial, en la siguiente tabla: Tabla 3. Comunalidades Tabla 3. Comunalidades Tabla 3. Comunalidades C. Total Varianza explicada Los autovalores iniciales expresan la cantidad de varianza total que está explicada por cada componente o factor. De esta forma, se obtuvieron cuatro (4) valores mayores a uno (1) que explican el 62.184% de la varianza total. En este caso se sugiere retener 4 componentes. Se aplica criterio de Kaiser, que consiste en conservar aquellos factores cuyo autovalor asociado sea mayor que 1, según la tabla a continuación: Tabla 4. Explicación del Total de la Varianza Tabla 4. Explicación del Total de la Varianza D. Gráfico de sedimentación. De igual forma, se genera el gráfico de la varianza que está asociado a cada factor, el cual nos permite determinar cuántos factores se deben conservar. En el caso de estudio, se considerarán los componentes 1, 2, 3 y 4, validando el método de Kaiser aplicado anteriormente. 48 Alviarez. Análisis multivariante del impacto e interdependencia del teletrabajo con variables de productividad, eficiencia, eficacia, satisfacción laboral y conocimiento en herramientas digitales: un caso de estudio. 48 Minerva Journal Vol.3, Issue. 8, (pp. 42-53) ISSN-E: 2697-3650 Fig. 4. Gráfico de sedimentación Se puede observar en el gráfico anterior que la trama muestra una ruptura distinta entre la pendiente pronunciada de los factores grandes y la salida gradual del resto. Fig 4 Gráfico de sedimentación Fig. 4. Gráfico de sedimentación Fig. 4. Gráfico de sedimentación Se puede observar en el gráfico anterior que la trama muestra una ruptura distinta entre la pendiente pronunciada de los factores grandes y la salida gradual del resto. E. Matriz de Componentes Principales: La Matriz de Componentes, Matriz de Cargas o Saturaciones Factoriales, señala la carga de cada variable en cada factor, de forma que los factores con pesos factoriales más elevados en términos absolutos indican una relación estrecha con las variables. De esta forma, la siguiente matriz presenta la solución factorial, la cual está constituida por las correlaciones entre las variables originales o saturaciones para cada componente o factor. Tabla 5. Matriz de Componentes Principales Tabla 5. Matriz de Componentes Principales 49 Alviarez. Análisis multivariante del impacto e interdependencia del teletrabajo con variables de productividad, eficiencia, eficacia, satisfacción laboral y conocimiento en herramientas digitales: un caso de estudio. 49 Minerva Journal Vol.3, Issue. 8, (pp. 42-53) ISSN-E: 2697-3650 Método de extracción: Análisis de Componentes Principales Método de extracción: Análisis de Componentes Principales a. Comunalidades reproducidas. b. Los residuos se calculan entre las correlaciones observadas y reproducidas. Hay 19 (52,0%) residuos no redundantes con valores absolutos superiores a 0,05. F. Matriz de correlaciones resultantes 42-53) ISSN-E: 2697-3650 -Representación tridimensional de los Componentes Principales: A partir del procesamiento del análisis de componentes principales se obtuvo la siguiente graficación: -Representación tridimensional de los Componentes Principales: A partir del procesamiento del análisis de componentes principales se obtuvo la siguiente graficación: Fig. 5, Representación tridimensional de los componentes principales Fig. 5, Representación tridimensional de los componentes principales F. Matriz de correlaciones resultantes -Test de esfericidad de Barlett. Para comprobar que las correlaciones entre las variables son distintas de cero de modo significativo, se comprueba si el determinante de la matriz es distinto de uno, es decir, si la matriz de correlaciones es distinta de la matriz unidad. - La medida Kaiser-Meyer-Olkin de adecuación del muestreo obtenido en el caso fue 0.553 está entre 0.75 y 0.5. por lo que es aceptable, tal y como se visualiza en la siguiente tabla: Tabla 6. KMO & Brlett´s Test Tabla 6. KMO & Brlett´s Test Como p (el nivel de significancia) es menor que 0,05 se rechaza la Hipótesis nula, donde se expresa la matriz de correlación es una matriz de identidad. De esta forma, se acepta la Hipótesis alternativa: el modelo de factor es apropiado Tabla 6. KMO & Brlett´s Test Como p (el nivel de significancia) es menor que 0,05 se rechaza la Hipótesis nula, donde se expresa la matriz de correlación es una matriz de identidad. De esta forma, se acepta la Hipótesis alternativa: el modelo de factor es apropiado. -Reproducida. Representa la matriz de las correlaciones que se obtiene a partir de la solución factorial hallada. A continuación, se presenta la tabla con los resultados de la correlación reproducida, donde se logra obtener un diagnóstico bueno del modelo y el número de factores el adecuado, ya que la estructura factorial fue capaz de reproducir la matriz de correlaciones, en función de las comunalidades finales. Tabla 7. Correlación Reproducida Tabla 7. Correlación Reproducida munalidades reproducidas. residuos se calculan entre las correlaciones observadas y reproducidas. Hay 19 (52,0%) residuos no redundantes con es absolutos superiores a 0,05. a. Comunalidades reproducidas. b. Los residuos se calculan entre las correlaciones observadas y reproducidas. Hay 19 (52,0%) residuos no redundantes con valores absolutos superiores a 0,05. 50 Alviarez. Análisis multivariante del impacto e interdependencia del teletrabajo con variables de productividad, eficiencia, eficacia, satisfacción laboral y conocimiento en herramientas digitales: un caso de estudio. 50 -Representación tridimensional de los Componentes Principales: A partir del procesamiento del análisis de componentes principales se obtuvo la siguiente graficación: ISSN-E: 2697-3650 Minerva Journal Vol.3, Issue. 8, (pp. 42-53) Minerva Journal Vol.3, Issue. 8, (pp. G. Propuestas de Etiquetas referenciales de los Nuevos Componentes. 1er Componente, es un factor que vincula la variable Eficacia con la Productividad. 2do Componente, es un factor que vincula Conocimientos de herramientas digitales con el nivel de satisfacción y el nivel laborales. 1er Componente, es un factor que vincula la variable Eficacia con la Productividad. 3er Componente, es un factor que integra antigüedad, con la edad y el grado laboral. 4to Componente vinculado a la eficiencia según objetivos alcanzados 3er Componente, es un factor que integra antigüedad, con la edad y el grado laboral. 3er Componente, es un factor que integra antigüedad, con la edad y el grado laboral. 4to Componente, vinculado a la eficiencia según objetivos alcanzados. 3er Componente, es un factor que integra antigüedad, con la edad y el 4to Componente, vinculado a la eficiencia según objetivos alcanzados. 4to Componente, vinculado a la eficiencia según objetivos alcanzados. Posteriormente, se desarrolla un análisis de regresión de la variable Teletrabajo sobre los tres factores o componentes principales. Para ello, se utiliza el software SPSS y se obtiene el resumen del modelo, donde se obtiene el estadístico Durbin-Watson superior a 1, lo que implica la no autocorrelación entre factores: Tabla 8. Resumen del Modelo Se logra obtener un estadístico de Durbin-Watson mayor a 1: 1,387 Tabla 9. Anova del Modelo Tabla 8. Resumen del Modelo Se logra obtener un estadístico de Durbin Watson mayor a 1: 1 387 Se logra obtener un estadístico de Durbin-Watson mayor a 1: 1,387 Tabla 9. Anova del Modelo Tabla 9. Anova del Modelo Tabla 9. Anova del Modelo 51 Alviarez. Análisis multivariante del impacto e interdependencia del teletrabajo con variables de productividad, eficiencia, eficacia, satisfacción laboral y conocimiento en herramientas digitales: un caso de estudio. 51 Minerva Journal Vol.3, Issue. 8, (pp. 42-53) ISSN-E: 2697-3650 Tabla 10. Coeficientes Se propone la siguiente ecuación final: Se propone la siguiente ecuación final: Variable Teletrabajo = 54.549 + 6.392(factor score1) – 3.266 (factor score2) – 0.262 (factor score3) + 0.457(factor score 4). CONCLUSIONES A través de la aplicación de las técnicas de análisis multivariante interdependiente: Análisis de Componentes Principales y Análisis factorial, se lograron obtener los 04 componentes principales con una varianza total de 62.184% (mayor a 60%), bajo las siguientes etiquetas: 1er Componente, es un factor que vincula la variable Eficacia con la Productividad. 2do Componente, es un factor que vincula Conocimientos de herramientas digitales con el nivel de satisfacción laboral y el nivel. 3er Componente, es un factor que integra Antigüedad, con la edad y el grado laboral. 4to Componente, vinculado a la eficiencia según objetivos alcanzados. Se presentaron los resultados de los descriptivos estadísticos: la media, la desviación estándar y el número de casos válidos para cada variable. KMO: arrojó aceptación de las variables con 0.553% (aceptable entre 0.5 y 075). La Prueba de esfericidad de Bartlett: Como p es menor que 0,05 se rechaza la Hipótesis nula, donde se expresa la matriz de correlación es una matriz de identidad. De esta forma, se acepta la Hipótesis alternativa: el modelo de factor es apropiado. En la diagonal de la matriz reproducida se encuentran las Comunalidades finales. La matriz de las correlaciones que se obtiene a partir de la solución factorial hallada resultó demostrar que el modelo es aceptable y el número de factores el adecuado, ya que la estructura factorial fue capaz de reproducir la matriz de correlaciones junto con las comunalidades finales. Finalmente, partiendo de la Regresión lineal y vinculación de la variable teletrabajo con los componentes nuevos se obtiene la ecuación de teletrabajo: Teletrabajo = 54.549 + 6.392(factor score1) – 3.266 (factor score2) – 0.262 (factor score3) + 0.457(factor score 4). 52 52 Alviarez. Análisis multivariante del impacto e interdependencia del teletrabajo con variables de productividad, eficiencia, eficacia, satisfacción laboral y conocimiento en herramientas digitales: un caso de estudio. Minerva Journal Vol.3, Issue. 8, (pp. 42-53) Minerva Journal Vol.3, Issue. 8, (pp. 42-53) ISSN-E: 2697-3650 REFERENCIAS [1] D. Colombo, «sedici.unlp.edu.ar,» Universidad Nacional de La Plata, 2008. [En línea]. Available: http://sedici.unlp.edu.ar/handle/10915/1933. [Último acceso: 9 Enero 2021]. [2] TLAC Toyota, «Movility Toyota,» Argentina, 2020. [2] TLAC Toyota, «Movility Toyota,» Argentina, 2020. [3]«scielo.org.co,» Universidad del Norte, 2013. [En línea]. 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https://openalex.org/W4376058116	https://unissa.edu.bn/journal/index.php/jhst/article/download/455/459	English	null	Halal Food Producer as An Alternative Employment Job for Halal Science Graduates in Brunei Darussalam	Journal of halal science and technology	2,022	cc-by	6,741	Journal of Halal Science and Technology (2022), 1(1): 55-66 ISSN 2957-4188 \| eISSN: 2957-4196 Journal of Halal Science and Technology (2022), 1(1): 55-66 ISSN 2957-4188 \| eISSN: 2957-4196 55 ABSTRACT Employment has always been a source of worry among university graduates. The unemployment rate is possibly the most well-known labour market indicator and certainly one of the most regularly mentioned by the media in many countries, as it is thought to best reflect the lack of employment at the national level. In nations that collect data on the labour force, it gives the broadest measure of the labour market situation, along with the employment to population. A quantitative survey was conducted to scrutinize how halal science graduates could get themselves employed in the halal food sector. The survey questionnaires were designed and targeted at those who venture into the halal food business. The study resulted that the halal food producer may gain the lowest and highest monthly income of less than five hundred and more than five thousand respectively. Thus, with the good marketability of halal food and resources, there is a vast opportunity for these halal graduates to venture into businesses selling halal food as they can implement all knowledge; they gained in their halal science studies at the University. Keywords: Employment Opportunities, Graduates, Halal Food Producer, Halal Science HALAL FOOD PRODUCER AS AN ALTERNATIVE EMPLOYMENT JOB FOR HALAL SCIENCE GRADUATES IN BRUNEI DARUSSALAM Nurdeng Deuraseh, Norkhairiah Hashim, Raihana Mohd Raffi, Nurul Jannahtul Hanani Rosli Halalan Thayyiban Research Centre, Universiti Islam Sultan Sharif Ali, Negara Brunei Darussalam. Nurdeng.deuraseh@unissa.edu.bn. Halalan Thayyiban Research Centre, Universiti Islam Sultan Sharif Ali, Negara Brunei Darussalam. *Nurdeng.deuraseh@unissa.edu.bn. Introduction Amongst the most pressing matters in Brunei Darussalam at present is securing stable jobs for university graduates who have completed the courses they have undertaken. The matter is related to Brunei Darussalam National Strategy called Wawasan Brunei 2035, which calls for the country to act and plan – to be renowned globally for the accomplishments of its well- educated and highly competent citizens. The Brunei government has made various attempts to attain Wawasan 2035, including offering free education to its citizens (Wawasan Brunei 2035, 2019). International Monetary Fund (IMF) 2019 country report stated that Brunei has the highest youth unemployment rate among ASEAN nations, and the survey conducted by the Labor Force Department, they have identified the unemployment rate has increased tremendously from 6.9 percent to 9.3 percent in 2014 to 2017 respectively (Ministry of Finance and Economy, 2019). Teichler (1996) emphasized that there are three main problems of ‘over education and the mismatch between demand and supply; the wide range of higher education and employment opportunities, the increase in graduate unemployment, and risky employment. These problems are related to the current situation of employment in this country. A study on unemployment issues among registered job seekers for both graduates and non-graduates conducted by Cheong and Lawrey (2009) revealed that there is a major mismatch between skills and desire for employment, as well as severe incompatibility of job expectations and actual responsibility in the job market. Moreover, the private sector is having difficulty in attracting the local skilled workforce as the job market has always been dominated by the government sector. Journal of Halal Science and Technology (2022), 1(1):55-66 56 Referring to the issues stated above, this paper explores the employment opportunity in the halal sector and its necessary traits. Universiti Islam Sultan Sharif Ali has established the Halalan Thayyiban Research Centre in 2018 in response to the Titah of His Majesty during the convocation ceremony of UNISSA in 2016. This Centre offered several courses for undergraduate and postgraduate students. One of the postgraduate courses available is halal science in which the students are expected to understand that halal discipline comprises a lot of aspects in Muslim’s daily basis, this includes halal food, halal education, halal tourism, halal finance, halal logistics, halal cosmetics, halal pharmaceuticals, and many more that associated with the application of science and technology in this contemporary era. Introduction Nevertheless, this paper will be focusing on how halal food producers can be a bright employment opportunity for the graduates in providing themselves with job security. Halal food has dominated the halal market as food is a necessity for everyone to stay alive and needs to be consumed every day and then (Zainal and Abdullah, 2018). 1. Literature Review 1.1. Definition of Halal Halal is rooted in an Arabic word, it defines as permissible or legal according to Shariah Law, while the opposite of halal is haram, which means prohibited or forbidden. In addition to halal, thayyib is defined as clean, wholesome, and beneficial. Islam has taught all Muslims that all types of food and drinks are halal except those considered prohibited. Muslims must consume halalan thayyiban food as commanded by Allah SWT through a few Quranic verses and hadith such as: a) Al-Baqarah, verse 68 which translates: “O Mankind! Eat of what is lawful and clean on the earth and do not follow the footsteps of Shaitan, indeed to you he is a clear enemy.” “O Mankind! Eat of what is lawful and clean on the earth and do not follow the footsteps of Shaitan, indeed to you he is a clear enemy.” b) Al-Maidah, verse 88 which translates: b) Al-Maidah, verse 88 which translates: “And eat of what Allah has provided for you (which is) lawful and good. And fear Allah, in whom you are believers.” c) It is reported by Bukhari, Muslim, Abu Daud, and Ibn Majah that the Prophet Muhammad SAW said: “What is Halal is clear. And what Haram is also clear. And in between those two is a dubious area which many people do not know about. So, whoever distanced himself from it, has acquitted himself (from blame). And those who fall into it, he has fallen into a state of Haram”. Deuraseh, N., et.al \| Halal Food Producer as An Alternative Employment Job for Halal Science Graduates in Brunei Darussalam 1.2. Halal Food Halal food and drink comprised all substances used are halal, halal on how to process the food, halal on how to get it and this include non-halal drinks as well (Deuraseh, 2019). Thus, halal food is classified into a few aspects: Halal food and drink comprised all substances used are halal, halal on how to process the food, halal on how to get it and this include non-halal drinks as well (Deuraseh, 2019). Thus, halal food is classified into a few aspects: a) Halal food substances. Allah SWT is indeed the Most Gracious, He has provided us with countless types of food that are halal to be consumed. Yet, He only prohibits very few types of food that cannot be eaten by Muslims. The wisdom behind the prohibition is no other than for the benefit and good welfare of humanity itself. Allah SWT has firmly Journal of Halal Science and Technology (2022), 1(1): 55-66 57 forbidden carcasses, blood, and swine. This includes all animals that did not die or were slaughtered by the Shariah Law. forbidden carcasses, blood, and swine. This includes all animals that did not die or were slaughtered by the Shariah Law. b) Halal on how to process the food. There is always a possibility that the food will not be processed in a Halal way which will make the food becomes unclean and unlawful for consumption. Islam has set a few conditions for halal animal slaughtering that need to be fulfilled; must be done by a Muslim slaughterer who owns a halal slaughtering certificate, must use a sharp knife to slit the throat of animals preferably with a single swipe, and make sure to protect the welfare of the animals. These animals might indirectly and unintentionally able to cause contamination by materials that are not halal throughout the process. c) Halal on how to get the food. A good Muslim will always strive his best to only consume halalan thayyiban food and drink that is spiritually healthy and hygienic. If the food is consumed unlawfully, the food will become the flesh and blood that will flow throughout the body which will negatively affect the spirituality of the consumer c) Halal on how to get the food. A good Muslim will always strive his best to only consume halalan thayyiban food and drink that is spiritually healthy and hygienic. 1.2. Halal Food If the food is consumed unlawfully, the food will become the flesh and blood that will flow throughout the body which will negatively affect the spirituality of the consumer d) Prohibition of Non-Halal drinks. All types of intoxicating drinks are prohibited. This includes drinks that are tainted with intoxicants or non-halal ingredients even with very little amount. In this contemporary era, these drinks have been distributed in the form of alcoholic drinks. d) Prohibition of Non-Halal drinks. All types of intoxicating drinks are prohibited. This includes drinks that are tainted with intoxicants or non-halal ingredients even with very little amount. In this contemporary era, these drinks have been distributed in the form of alcoholic drinks. It is also noted halal products must be free from prohibited elements as mentioned in the Quran (Raffi, 2019). Additionally, according to Brunei Standard for Halal Food PBD 24:2007, halal food means food fit for human consumption and permitted by hukum syara’ and fulfils the following conditions (Deuraseh, 2019): It is also noted halal products must be free from prohibited elements as mentioned in the Quran (Raffi, 2019). Additionally, according to Brunei Standard for Halal Food PBD 24:2007, halal food means food fit for human consumption and permitted by hukum syara’ and fulfils the following conditions (Deuraseh, 2019): a) The food or its ingredients that do not contain any parts or products or animals that are non-halal to Muslims according to hukum syara’ or products of animals which are not slaughtered according to hukum syara’. b) The food does not contain any ingredients that are najis(impurities) according to hukum syara’. The food that is safe and not harmful. c) The food that is safe and not harmful. d) The food that is not prepared, processed, or manufactured using equipment that is contaminated with things that are najis(impurities) according to hukum syara’. e) The food or ingredients do not contain any human parts or its derivatives that are not permitted by hukum syara’. f) During its preparation, processing, packaging, storage, or transportation, the food is physically separated from any other food that does not meet the requirements stated in terms of (a), (b), (c), (d), or (e) or any other things that have been decreed as najis(impurities) by hukum syara’. Deuraseh, N., et.al \| Halal Food Producer as An Alternative Employment Job for Halal Science Graduates in Brunei Darussalam 1.3. Halal Food Producer Halal food producer is people who make and supply Halal food for consumption. They are responsible for protecting the permissibility and hygienic aspect of food from raw materials to the point of consumers’ consumption. (W. Sari, 2014, Roziah Atan 2018) has listed several producers’ behaviours by following Islamic values which need to be implemented: a) Long-term insight, oriented towards the afterlife goal. a) Long-term insight, oriented towards the afterlife goal. Deuraseh, N., et.al \| Halal Food Producer as An Alternative Employment Job for Halal Science Graduates in Brunei Darussalam Darussalam Journal of Halal Science and Technology (2022), 1(1):55-66 58 b) Keeping promises and contracts, both in the internal or external scope. c) Meet the measurements, accuracy, accuracy, and truth; d) Sticking to discipline and dynamic. d) Sticking to discipline and dynamic. e) Glorify achievement/productivity. f) Encouraging ukhuwah between economic actors. g) Respecting individual property rights. h) Following legal contract/transaction requirements. i) Fair in transactions. j) Having social insight. k) Payment of wages on time and reasonable. l) Avoiding types and production processes are forbidden in Islam. Deuraseh, N., et.al \| Halal Food Producer as An Alternative Employment Job for Halal Science Graduates in Brunei Darussalam 1.4. Good Manufacturing Practice Deuraseh, N., et.al \| Halal Food Producer as An Alternative Employment Job for Halal Science Graduates in Brunei Darussalam Darussalam Journal of Halal Science and Technology (2022), 1(1): 55-66 59 g) Recall and traceability: methods used to ensure final products are correctly coded and labelled. All of the incoming and outgoing materials are traceable; the recall system must be set up properly and tested for its effectiveness. g) Recall and traceability: methods used to ensure final products are correctly coded and labelled. All of the incoming and outgoing materials are traceable; the recall system must be set up properly and tested for its effectiveness. h) Water safety: guidelines to inspect include water, ice, and steam as well as water treatment standards to ensure it is safe for food processing. h) Water safety: guidelines to inspect include water, ice, and steam as well as water treatment standards to ensure it is safe for food processing. GMPs comprised of two main components that are important in achieving a good food safety system: a) Written programs are all mandatory policies, guidelines, and procedures that need to be ensured to meet the standards listed in each GMP program. These includes: a) Written programs are all mandatory policies, guidelines, and procedures that need to be ensured to meet the standards listed in each GMP program. These includes: i. Policies and procedures: standard operating procedures (SOPs) and sanitation standard operating procedures (SSOPs) ii. Monitoring procedures. ii. Monitoring procedures. iii. Recordkeeping: methods to keep and handle the records. b) Implementation comprises conducting GMPs related activities and monitoring can be done accordingly. There shall be sufficient and appropriate education, training, and skill regarding the plant operations that can be provided to everyone who is working in the unit. The organization must highlight the critical points where food safety can be compromised. Several factors will affect the types and level of training (Manning & Baines, 2004): There shall be sufficient and appropriate education, training, and skill regarding the plant operations that can be provided to everyone who is working in the unit. The organization must highlight the critical points where food safety can be compromised. Several factors will affect the types and level of training (Manning & Baines, 2004): a) The source of the food, specifically its ability to resist the growth of pathogenic or spoilage microorganisms. 1.4. Good Manufacturing Practice b) The technique of handling, packing and reducing the possibility of contamination of the food. b) The technique of handling, packing and reducing the possibility of contamination of the food. c) Extensive food preparation before its final consumption. c) Extensive food preparation before its final consumption. d) The storage conditions of the food. d) The storage conditions of the food. e) The shelf life of the food must be informed before consumption. 1.4. Good Manufacturing Practice Good Manufacturing Practices (GMPs) are the fundamental operational and environmental conditions required to ensure safety throughout the production processes (Raffi, 2019). The Food and Drug Administration (FDA) of the United States of America has introduced and enforced manufacturing guidelines that should be applied and performed by food producers that can affect both the safety and quality of food products. GMPs cover extensive guidelines ranging from employees, equipment, process, and condition of its surrounding in the production process. Below are several criteria that GMPs have addressed: a) Environmental control: location, design, the structure of the building, its interior, equipment, and water supply of the premises. a) Environmental control: location, design, the structure of the building, its interior, equipment, and water supply of the premises. b) Personnel practices: All personnel involved throughout the production processes need to implement good hygiene, as it will give an effect on food safety and quality. Any person who entered the place where food is being prepared needs to ensure that they do not contaminate the food product. They also need to comply with the standards of procedure of food production; hand washing, proper clothing, using footwear, and headwear. c) Shipping, receiving, handling, and storage: inspection procedures for transport vehicles, loading, unloading, and storage practices, inspection procedures for incoming products, shipping conditions, returned and defective products, allergen control, chemical storage, and waste management. d) Pest control: always monitor both exterior and interior aspects of the building and apply pesticides. e) Sanitation: methods of sanitizing and cleaning as well as pre-operational evaluation. f) Equipment maintenance: guidelines for preventive maintenance as well as calibration of all equipment and instruments that can affect food safety. This includes but is not limited to thermometers, thermocouples, metal detectors, scales, and pH meters. f) Equipment maintenance: guidelines for preventive maintenance as well as calibration of all equipment and instruments that can affect food safety. This includes but is not limited to thermometers, thermocouples, metal detectors, scales, and pH meters. f) Equipment maintenance: guidelines for preventive maintenance as well as calibration of all equipment and instruments that can affect food safety. This includes but is not limited to thermometers, thermocouples, metal detectors, scales, and pH meters. Deuraseh, N., et.al \| Halal Food Producer as An Alternative Employment Job for Halal Science Graduates in Brunei Darussalam Deuraseh, N., et.al \| Halal Food Producer as An Alternative Employment Job for Halal Science Graduates in Brunei Darussalam 1.5. Brunei Halal Certification Halal food producer is obliged to have a halal permit and halal logo for their business as per required by Halal Certificate and Halal Label Order 2005. This Order is known as the legal framework for Halal Certification in Brunei. Halal certificate and halal permit are compulsory for business premises such as restaurants or eateries and food products respectively. It is mandatory for food business owners in Brunei Darussalam to apply them. A halal certificate is compulsory for any businesses that involve food preparation that including restaurants, cafes, food courts, and central kitchens. Whereas halal permit is specifically used for food processing facilities such as factories. About the provision in the Order, there should be at least two halal supervisors on the business premises who are responsible to monitor halal-related matters concerning food and company. Brunei Halal logo has the most crucial element that the main authority controls the ratification of this logo. Brunei halal certification and halal logo are known to have a stringent process of acquiring and this is the reason why Brunei halal logo is very well trusted by other international certifying bodies. Deuraseh, N., et.al \| Halal Food Producer as An Alternative Employment Job for Halal Science Graduates in Brunei Darussalam Journal of Halal Science and Technology (2022), 1(1):55-66 60 Figure 1: Flowchart for Application of Halal Certificate Source: Halal Food Control Division, Ministry of Religious Affairs Figure 1: Flowchart for Application of Halal Certificate Source: Halal Food Control Division, Ministry of Religious Affairs Deuraseh, N., et.al \| Halal Food Producer as An Alternative Employment Job for Halal Science Graduates in Brunei Darussalam 1.6. Marketability of Halal Food Muslims has made up a 1.9 billion worldwide population whereby most of them live-in South-East Asia, the Middle East, and North Africa. Their population will always be increasing and it is expected to conquer up to two-fifth of the world population. ASEAN countries such as Malaysia, Indonesia, and Singapore are considered big players in halal markets which are all located close to Brunei Darussalam. According to Thomson Reuter and Dinar Standard (2015), in 2014 it is estimated that global Muslim spending on food and drinks (F &B) increased 4.3% to reach $1.128 billion. This condition makes the potential of the halal food market reaches 16.7% of global spending in 2014 (Standard, 2015). Nonetheless, Hasan and Awang (2009) stated that Muslim minority countries such as Australia, Canada, France, and New Zealand have been trying to produce halal food as they realized that there is a good opportunity to tap into the Halal market. They are also well aware of the significance of halal business, as the halal niche market will essentially contribute to their country’s revenue (Deuraseh & Heradhyaksa, 2020). Brunei Darussalam is an Islamic country in which 83 per cent of its population are Muslims. Hence, the halal industry is among the key players in diversifying the economy of this country. The halal food sector has been dominating the halal business which is commonly under the observation of Darussalam Enterprise (DARe). There are four types of businesses in Brunei; micro, small, medium, and enterprises (MSMEs). In response to this, DARe aims to assist local businesses and entrepreneurs to achieve dynamic and resilient company growth. With this, developmental programs for MSMEs which is known as Journal of Halal Science and Technology (2022), 1(1): 55-66 61 Industry Business Academy (IBA) have been introduced by DARe to escalate the growth of the local businesses (DARe, 2018). Industry Business Academy (IBA) have been introduced by DARe to escalate the growth of the local businesses (DARe, 2018). Industry Business Academy (IBA) have been introduced by DARe to escalate the growth of the local businesses (DARe, 2018). In an article published by bizbrunei.com in 2016, Brunei’s Halal industry generates $88 million in revenue and employed 835 people according to data reported by 35 companies. 1.6. Marketability of Halal Food The main contributor is the halal-certified food products, especially the products under the Brunei Halal brand that is run by the state-owned Ghanim International Corporation which has made an entity to the United Kingdom market as per shared by the Minister of Energy and Industry. He urged local micro, small and medium enterprises (MSMEs) to collaborate with other companies whether to manufacture, brand, market, or distribute their products in the domestic, regional, and even going for the global market. The Minister highlighted that the halal industry is expanding that he also touched upon the extensive and comprehensive principle of Halalan Thayyiban. This covers high values of Islamic law as well as adhering to maqasid shariah which aims to ensure that the “entire value chain” can provide the “highest standard”. The government has reduced the time in obtaining halal certification of food products from more than six months to 45 calendar days to ease the food producers and at the same time keep the efficiency and transparency of the process. Brunei Halal Foods brand is one of the distinctive government projects that is managed by a company named Ghanim International Corporation Sdn Bhd. The main objective of this brand is to be a platform for the development of local products and MSMEs. This project offers local MSMEs a good opportunity to manufacture their halal products and assists them to commercialise their products in the global market through Ghanim International Corporation Sdn Bhd. Most importantly, their objectives include; fulfilling the obligation of fardhu kifayah (a legal obligation that must be discharged by the Muslim community as a whole) in Islam which is the Muslims’ duty to facilitate the supply of halal products to Muslims around the world (Haji Mustafa, 2009). Due to the high integrity of ‘Brunei Halal Foods’ brand in terms of repacking products, marketing, and distribution, it enables them to market the halal products efficiently and effectively to regional markets and Gulf Coast countries, as well as Europe and the United States (Fleishman- Hillard Majlis, 2011). Moreover, this brand is very confident in assuring the consumers that products manufactured under its brand have gone through the best security, and standards check and have been tested to meet and exceed the expectations of halal consumers as their main goal is to provide high-quality products to the global halal market. Deuraseh, N., et.al \| Halal Food Producer as An Alternative Employment Job for Halal Science Graduates in Brunei Darussalam 1.6. Marketability of Halal Food Global collaboration between Brunei Meat Company (BMC) and four Japanese Companies aims of bringing in Brunei-Made Halal products into the Japanese market by next year (Kon, 2019). The collaboration took years of discussion, product-testing between the companies involved and the endless efforts of the Economic Research Institute for ASEAN and East Asia (ERIA) which has accomplished its main goal of bringing interested parties together. Okuochi Hiroshi is an advisor who has said to the President for Brunei Affairs of ERIA; “Brunei Halal is a premium certification for Halal in the world, based on a report on the Study on Halal Industry Investment in Brunei Darussalam carried out by ERIA in 2016”. Soon after the Halal Seminar report is released, the four Japanese companies namely Chibo, Ginrei Service Co Ltd, Kanda Farm Co, and Harry’s were very interested in the Brunei Halal Certification and visited Brunei to look for collaboration potential. The Chibo company has come up with a Brunei-made and Halal certified okonomiyaki from Brunei Meat Company and there will be more products to be introduced together with the other three companies. Okuochi even mentioned that the products will be exported to Indonesia and Malaysia and they will be supplied to the 2020 Tokyo Olympic Games and Deuraseh, N., et.al \| Halal Food Producer as An Alternative Employment Job for Halal Science Graduates in Brunei Darussalam Journal of Halal Science and Technology (2022), 1(1):55-66 62 Paralympic Games. This is just an indication of how strong the brand of Brunei Halal is in the eyes of other countries. Paralympic Games. This is just an indication of how strong the brand of Brunei Halal is in the eyes of other countries. In this regard, with great support from the government, there is no doubt that halal food products have high potential both in the local and global markets. Hence, the next part of this paper will try to look at how the current local halal food businesses have been doing so far and whether being a halal food producer can be a stable career for the halal graduates. 2. Materials and methods To gather the necessary data, the instrument used to collect data in this research is a questionnaire. According to Chua (2006), a questionnaire is a set of written questions in the respondents are required to answer based on the options or alternatives given. 10 questionnaires were distributed to a large number of people with diverse backgrounds, perspectives, and interpretations of Halal implementation, government rules and regulations, and consumer acceptance of Brunei gaining accreditation widely and globally, allowing and leading to better data collection from various perspectives. The method used for data collection of this research is a quantitative analysis. There are 14 questions altogether in this survey by which all of which are specifically designed and targeted at those who already own the halal food business. Survey questionnaires were made using Google Forms and distributed through online platforms via WhatsApp, Instagram, and Facebook Messenger. Deuraseh, N., et.al \| Halal Food Producer as An Alternative Employment Job for Halal Science Graduates in Brunei Darussalam 3. Results and Discussion 3. 3. Meanwhile, 36.7% of the respondents are selling savouries and including rice, meats, and pasta. The rest of the respondents are selling snacks-type foods. Bruneians are known to favour sweet-tasting food and this shows there is a high market for sweet treats than any other type of food. The data reveals there are only 2 of the respondents (6.6%) have applied for the Brunei halal certificate. Halal certification is crucial in halal food, based on this data there are still a lot of these food sellers who have not yet applied for the halal certification which may be due to the thought of they are only doing it part-time. Presumably, a few things may hinder these food sellers from applying for halal certification such as; the stringent process, high cost of application, and the requirement of getting separate places to make the foods that are to be sold to others. Aside from collecting information for job employment, the purpose of this question is to know what triggers these respondents to start making and selling foods in the first place. 21 respondents (73.3%) started their businesses as a passion to be involved in the halal food industry and the rest of them are either started doing food business because the respondents were eager to try out new things and while waiting to get a stable-permanent job. Having passion is essential in building a business because it is an inner strength that will push them to be successful. With passion, it inspires the vision for the business even if it is in a niche that no one has done before, it helps people to set a solid foundation for their business and establish core values, and it helps people to create authentic content that resonates with right customers as well setting them apart from other competitors. However, it can be seen that some of the respondents are selling foods out of curiosity in trying new things and to get income while waiting for a permanent job, this shows how the people are more open-minded and do not solely rely on the government to provide jobs for them. The finding shows that, in profit-making, 17 respondents (56.7%) earn less than $500, while 8 respondents (26.7%) earn between $500 to $1000, and 5 respondents (16.7%) $1500. This profit is calculated by their monthly earnings through business. 3. A survey was done and collected from 30 respondents who had answered the questionnaires and all respondents are operators of online/home-based food businesses. For this study, the identity of the respondents is kept confidential to respect and protect their privacy. The survey result shows there are 10 respondents (33.3%) who make and sell food as their full-time job while the rest 20 respondents (66.7%) are only selling food as their part-time job. Predominantly in Brunei Darussalam, there are a lot of people who have a permanent job yet still find other side income. This might be due to their great passion for cooking and baking, or it might be due to an insufficient amount of salary to cover their current living expenses. Nonetheless, both modes of business have shown the respondents’ effort in getting profit that is halal and full of barakah (blessings). The survey question was also focusing on the period these food businesses have been established, and the result from the survey shows that more than half of the respondents have been in this industry for more than 5 years and the rest of them are those who just entered the market in less than 3 years. It has proven that the food business has a very high potential to be sustainable in the halal food industry. The result shows half of the respondents (15) are University graduates. Meanwhile, the rest of the respondents are postgraduate, diploma, and Brunei Cambridge GCE ‘O’ Level which takes up 26.7%, 20%, and 3.3% respectively. Education develops critical thinking. This is vital in teaching a person how to use logic when making decisions and interacting with people. With education, people tend to have better health and wellbeing, higher social trust, greater political interest, lower political cynicism, and less hostility towards immigrants. These traits are what made good-characters citizens who will be able to comply with any regulation given by the government. These people also tend to have a strong personality and are very optimistic that they will be able to overcome any struggles in life. Journal of Halal Science and Technology (2022), 1(1): 55-66 63 In terms of categories and types of food, the information and data show the result by which more than half of the respondents are selling sweet treats such as cakes, cookies, tarts, and other types of desserts. Deuraseh, N., et.al \| Halal Food Producer as An Alternative Employment Job for Halal Science Graduates in Brunei Darussalam 4. Conclusion and Recommendation This study discusses the prospect of halal food producers as alternative employment for halal science graduates. People do not have an open knowledge about halal, thus the demand for ‘halal’ employment is not as much. The reality is that there are no private companies that demand employees with halal science backgrounds now. Based on the analysis of the data collected through 30 respondents, currently, only the government is offering employment but with a limited number of hiring. Hundreds of students graduating in the halal study, and being halal food producers are indeed having the highest potential to get themselves employed immediately. With strong knowledge about halal, these graduates are obliged to implement everything they learned throughout the Halal courses in producing halal food in pursuance of meeting the Halal standards. Anyone who wants to start selling foods, they may seek help from DARe as their purpose is to help the new entrepreneurs grow efficiently by providing systematic approaches and developmental courses. Initiating collaboration with other companies such as Ghanim International Corporation for the production and marketing of halal food products will be a good step in exposing their products to potential customers. In addition, those who are willing to invest more in their halal food business may enter the halal global market as the halal industry is vastly growing worldwide. Apart from that, the respondents have given several recommendations for those who want to enter the halal food industry as they have more experience. In business, is vital to set the intention right to succeed with a good purpose. They mentioned that perseverance, tawakkal and always being kind to the customers no matter how difficult the situation can be, are among the things that must be implemented. Finally, they even emphasized that being a Halal food producer might not be as easy as it seems to be, but they need to set their goals straight and be persistent in this field. 3. On the other hand, these food sellers have acquired the highest amount of monthly income; 7 respondents (23.3%) of them have gained more than $5000 for their monthly income, 1 respondent has acquired its highest income of more than $3500, 10 respondents (33.3%) earned more than $2500, and 12 respondents (40%) earned their highest income of more than $1500. Based on this number, it can be observed that more than half of those who have the lowest income is less than $500 are only doing it part-time, while almost all of the respondents who earn their lowest-income of less than $1500 are doing it full time. Furthermore, some of these food sellers even gained more than $5000 per month; this amount is believed to be so much higher than the average salary of fresh graduates. The educational background of the respondents shows that they have no prior business knowledge, however, for the benefit of their business, several marketing steps and strategies have been taken to advertise their business. Marketing plays a vital role in introducing food products to potential customers. The results above have shown that all of the respondents have been using advanced technology and social media such as Instagram, Facebook, and WhatsApp to market their products. This just shows how technology has been dominating the world and using them to expand one’s business. Deuraseh, N., et.al \| Halal Food Producer as An Alternative Employment Job for Halal Science Graduates in Brunei Darussalam Journal of Halal Science and Technology (2022), 1(1):55-66 64 5. 5. The authors would like to thank this work is ostensibly supported by Universiti Islam Sultan Sharif Ali under the research grant entitled “Potential Employment opportunities in Halal Industry for UNISSA Halal Science Graduate Students from Stakeholders Perspective” – it was during this research period that the hypothesis presented herein was initially conjectured. Deuraseh, N., et.al \| Halal Food Producer as An Alternative Employment Job for Halal Science Graduates in Brunei Darussalam References Cheong, D., Lawrey, R. (2009). A study of unemployment issues among registered job seekers in Brunei Darussalam. Gadong: Centre for Strategic and Policy Studies (Brunei) Chua, Y.P. (2006). “Kaedah Penyelidikan.” Malaysia: McGraw-Hill. Deuraseh, N. (2019). Shariah and Life: Halalan Thayiban Perspective. Gadong: UNISSA Press. Deuraseh, N., Heradhyaksa, B. (2020). Potential Occupation in Halal Industry for Reinforcement Sharia Economic Law. 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https://openalex.org/W2600207793	https://newprairiepress.org/cgi/viewcontent.cgi?article=2062&context=fgr	English	null	Identification of b-galactosidases	Fungal genetics reports	1,969	cc-by-sa	649	Identification of b-galactosidases Identification of b-galactosidases H. N. Johnson Follow this and additional works at: https://newprairiepress.org/fgr This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Recommended Citation Recommended Citation Johnson, H. N. (1969) "Identification of b-galactosidases," Fungal Genetics Reports: Vol. 14, Article 31. https://doi.org/10.4148/1941-4765.2062 This Enzyme Methodology is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Fungal Genetics Reports by an authorized administrator of New Prairie Press. For more information, please contact cads@k-state.edu. Follow this and additional works at: https://newprairiepress.org/fgr s work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Fungal Genetics Reports Fungal Genetics Reports Fungal Genetics Reports Fungal Genetics Reports Volume 14 Volume 14 Article 31 Recommended Citation Recommended Citation Johnson, H. N. (1969) "Identification of b-galactosidases," Fungal Genetics Reports: Vol. 14, Article 31. https://doi.org/10.4148/1941-4765.2062 This Enzyme Methodology is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Fungal Genetics Reports by an authorized administrator of New Prairie Press. For more information, please contact cads@k-state.edu. This enzyme methodology is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/ This enzyme methodology is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol14/iss1/31 Abstract Abstract Identification of b-galactosidases Johnson, H. N. Identification of a third farm Strickland and Shields (1967 Neurorporo Nwrl. 12: 15) described o of D-goloctoridare by sectioning ocrylamide gels. method for rpecifically staining enzymes and matching them to proteins stained on the rome ocrylamide gel. This communication describer a more satisfactory methcd for identifying the R-golactosidoses in Neurcsporo. In addition to the previously described B-goloctoridorer with pH optima at 7.5 and 4.2, o third form has been identified b this methcd which has D pH optimum at 4.5. Johnson, H. N. Identification of a third farm of D-goloctoridare by sectioning ocrylamide gels. The rtondord 7.5% gels were used and after electrophorerir were frozen on dry ice or by immersion in liquid nitrogen. T h e were then split lengthwise with D razor blade and one half wets stained with amide block. The other half WOI then sectioned l-mm sections by a gel slicer. The slices were individually mroyed wing 0-nitro phenyl-R-D-gclloctopyranoside. The en- e activity for each farm of the enzyme uruolly WOI localized in 2 or 3 slices. Lking tracking dye (brom phenol blue) os D nt marker, Rf’r for the enzymes were fairly reproducible. Wild ‘ype Neurosporo crosso (74-OR-A) woz grown on 196 lactose for 5 days and the mycelio were filtered out on o &hoer - funnel. The mycelio were extracted with 10 ml of 0.01 M No phosphate, pH 7.5, per g of wet weight. The mycelio were home- genized in on Omni-Mixer, sonicoted, and then stirred for 2 hrs ot 4°C. After centrifugotion at 20,000 x g for 20 min, the super- noton+ war used os crude extract. The growth medium after filtration wm concentrated by dialysis ogointt dry sucro~c. Sampler for electrophoresis contained z 0.25 mg protein. Crude extract gave reoctiom with ONPG ot three distinct sites on the gel, The 7.5 enzyme hod on Rf of 0.046, the 4.2 en- zyme hod on Rf of 0.250, ond o third form of the enzyme hod on Rf of 0. 150. When the growth medium was electrophoresed, oc- tivity oppeored ot either one or two sites, depending upon the age of the culture. Medium from o young culture shaved only the new form of the enzyme (pH 4.5) w I e medium from on old culture contained both the 4.2 and 4.5 forms with o predominance h.1 of the former. Abstract Abstract Identification of b-galactosidases Between these two extremes there were gradations in the proportions of the two forms. This work supported in port by the NIH Training Grant in Genetics (TOI-GMOl316) to Florida State University. - - - Genetics Laboratories, Department of Biological Science, Florida State University, Tollohossee, Florida 32306.
https://openalex.org/W4392108375	https://www.frontiersin.org/articles/10.3389/feduc.2024.1326437/pdf?isPublishedV2=False	English	null	Teachers’ experiences of continuous professional development for citizenship and social cohesion in South Africa and Zimbabwe: enhancing capacity for deliberative democracies	Frontiers in education	2,024	cc-by	13,338	TYPE Original Research PUBLISHED 23 February 2024 DOI 10.3389/feduc.2024.1326437 TYPE Original Research PUBLISHED 23 February 2024 DOI 10.3389/feduc.2024.1326437 TYPE Original Research PUBLISHED 23 February 2024 DOI 10.3389/feduc.2024.1326437 COPYRIGHT © 2024 Singh and Mukeredzi. 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. Teachers’ experiences of continuous professional development for citizenship and social cohesion in South Africa and Zimbabwe: enhancing capacity for deliberative democracies OPEN ACCESS EDITED BY Jaime Ibáñez Quintana, University of Burgos, Spain REVIEWED BY Caroline Kuhn, Bath Spa University, United Kingdom Ajay Singh, University of Hail, Saudi Arabia CORRESPONDENCE Marcina Singh marcina.research@gmail.com; marcinas@uj.ac.za Marcina Singh1 and Tabitha Mukeredzi2 1SARChI Teaching and Learning, Faculty of Education, University of Johannesburg, Johannesburg, South Africa, 2School of Education, Faculty of Arts and Design, Durban University of Technology, Durban, South Africa Singh M and Mukeredzi T (2024) Teachers’ experiences of continuous professional development for citizenship and social cohesion in South Africa and Zimbabwe: enhancing capacity for deliberative democracies. While questions continue to be asked about teachers’ content and pedagogical content knowledge to ensure quality education systems, less consideration has been placed on teachers’ ability to teach for citizenship and social cohesion that contributes equally to quality education systems. This paper illuminates the understandings of citizenship and social cohesion held by South African and Zimbabwean teachers, their experiences of Continuous Professional Development (CPD) that promote the values of citizenship and social cohesion, and how they practice these learnings in their school contexts. The South African study presents the views of eleven high school teachers where data was procured through semi structured interviews. The Zimbabwean study presents the views of seventeen high school teachers, where data was procured through an open- ended questionnaire. The two studies suggest that teachers in South Africa and Zimbabwe share similar perspectives on citizenship and social cohesion, emphasizing nation-building and respect as key drivers. Teachers also report aligning their teaching practices with citizenship and social cohesion values with a limited focus on political participation, possibly due to fear of negative repercussions. Further, CPD for citizenship and social cohesion is fragmented, inconsistent and mostly absent. This study is an important contribution to debates about improving quality education and ensuring deliberative democracies in post-conﬂict and post-colonial states in the Global South. Teachers play a critical role in socializing schoolchildren for citizenship. As such, they need to be equipped with the skills that allow Introduction incorporate these values in their teaching practices and pedagogical approaches. The paper argues that equal emphasis should be placed on CPD for citizenship and social cohesion (and non-cognitive aspects in general) as this may support learners as they develop into adulthood to become participatory citizens who eﬀectively utilize their agency to build strong democracies. This is particularly important in countries, such as South Africa and Zimbabwe, where the lived reality is suggestive of a crumbling democracy. The positive eﬀects of good-quality teachers and teaching cannot be overstated. Teachers not only have the ability to improve the quality of education systems in general but, as agents of citizenship and social cohesion, they are able to mold and inﬂuence the actions and beliefs of learners as these youths embark on their journey to becoming participatory citizens in society (Araujo et al., 2016). Whilst there are several factors that may realize the objective of quality education, including school context, teaching and learning resources, working conditions and teacher governance, it is teacher training that is the most impactful ( Sayed et al., 2018). As a continuous process, the beginning of developing a teacher does not necessarily start at the onset of Initial Teacher Education (ITE), instead, teacher development starts when the teacher-to-be is still a learner in school. As such, given that what teachers do or do not do is in response to their early learning experiences (Allender and Allender, 2006). These beliefs are then sharpened, reinforced, or disrupted by formalized training. This paper is divided into seven sections. After the introduction, the paper commences with a discussion of the theoretical underpinnings of citizenship and social cohesion and how this often materializes in post-conﬂict and post- colonial states such as South Africa and Zimbabwe. This is followed by a situational analysis of CPD in the SADC region by discussing the latest CPD framework. The paper then discusses the provision of CPD in South Africa and Zimbabwe, with a speciﬁc emphasis on CPD for citizenship and social cohesion. The last three sections discuss the methodology, the ﬁndings, and the conclusions, respectively. This paper is concerned with the process of teacher training and development once students have graduated and have commenced their journeys as professionals. Training in this phase of teacher development is referred to as Continuous Professional Development (CPD), often interchanged in the literature as Continuous Professional Teacher Development (CPTD) or Teacher Professional Development (TPD). Introduction Popova et al. (2022, 108) note that “the principal tool that countries across the income spectrum use to improve the knowledge and skills of their practicing teachers is professional development (PD), which refers to on-the-job training activities ranging from formal, lecture-style training to mentoring and coaching.” Frontiers in Education KEYWORDS continuous professional development, citizenship, social cohesion, pedagogy, South Africa, Zimbabwe, deliberative democracy frontiersin.org Front. Educ. 9:1326437. doi: 10.3389/feduc.2024.1326437 COPYRIGHT © 2024 Singh and Mukeredzi. 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. 01 Frontiers in Education frontiersin.org Singh and Mukeredzi Singh and Mukeredzi 10.3389/feduc.2024.1326437 10.3389/feduc.2024.1326437 10.3389/feduc.2024.1326437 them to do so. Further to this, teachers also need the freedom and autonomy to discuss politics in the classroom without fear of negative repercussions, including alienation and fear of losing their jobs. Singh and Mukeredzi Citizenship and social cohesion as foundational to democracy Citizenship, social cohesion and democracy are interrelated, but interdependent for their full realization. They also operate at diﬀerent levels. Citizenship has a focus on individual rights, responsibilities and belonging (Marshall, 1950; Westheimer and Kahne, 2004; Yuval-Davis, 2006; Singh, 2020); social cohesion focuses on group dynamics (Durkheim, 1893; Rudiger and Spencer, 2004; Barolsky, 2016); and democracy operates at a societal level (Rawls, 1971). Cuellar (2009, 5) argues that “democracy and social cohesion promote the establishment of citizenship with rights and responsibilities diﬀerently but in a complementary manner.” Social cohesion is foundational to a stable democracy (Rawls, 1971) because it acknowledges the diversity of society, and promotes the values of citizenship, including the reduction of inequality. Whilst social cohesion can be deﬁned as “the extent of connectedness and solidarity among groups in society” (Manca, 2014, 6026), citizenship is about individual rights, responsibilities and belonging to those groups (Singh, 2020). As such, the link between social cohesion and citizenship is that “social cohesion refers to people’s relationships and interactions in society, including the role of citizenship” (Cuellar, 2009, 3). Citizenship and social cohesion together are required to realize a ﬂourishing democracy and are all In this paper, CPD is deﬁned as “activities that increase the knowledge and skill base of teachers” (Sayed and Bulgrin, 2020, 8). In drawing on empirical data from two studies, one in South Africa and one in Zimbabwe, this paper aims to ascertain teachers’ understanding of citizenship and social cohesion as well as teachers’ experiences of CPD for citizenship and social cohesion, and how these learnings are realized in practice. Social justice orientated, (see Rawls, 1971; Bell, 2007; Connell, 2014; De Sousa Santos, 2014), the ﬁndings suggest that, ﬁrstly, teachers’ understandings of citizenship and social cohesion are inﬂuenced by varied training experiences, opportunities and socio- political contexts. Secondly, these aﬀective elements of teaching and learning are rarely discussed and promoted through CPD programmes and, thirdly, despite very little exposure to CPD for citizenship and social cohesion, teachers still claim to promote and 02 frontiersin.org Singh and Mukeredzi 10.3389/feduc.2024.1326437 underpinned by the values of respect, inclusion, tolerance, equality, and recognition. Figure 1 below depicts the interrelated nature of citizenship, social cohesion, and democracy with their common embedded values. In countries where there is a history of violence and conﬂict, the pursuit of quality education is challenging when contexts remain shaped by inequality, exclusion, injustice and marginalization. Citizenship and social cohesion as foundational to democracy However, despite this, education remains the key medium through which social cohesion, social justice, citizenship and social solidarity can be mobilized and promoted therefore interventions that seek to realize this remain important (Durkheim, 1964). Social cohesion is increasingly recognized, in policy and academic discussions, as an important determinant of communities’ ability to absorb shocks, particularly in conﬂict and post-conﬂict aﬀected contexts, where limited state capacity often meets extensive urgent needs. South Africa and Zimbabwe are technical democracies in that their constitutions reﬂect states that subscribe to the values of democracy. However, in practice, the majority of their populations are not beneﬁtting from this, putting these countries and their citizens in disarray. South Africa and Zimbabwe are regional neighbors, both situated in sub-Saharan Africa, and they also share some historical similarities, such as being colonized by the British, leaving behind a legacy that impacted and still impacts language and the provision of education. Howell et al. (2018, 127) note that “the provision of education in both South Africa and Zimbabwe has been strongly shaped by the inequalities of their colonial pasts and the eﬀorts by their post-liberation governments to build new education systems where all children have equitable access to quality education.” In order to restore dignity, respect and recognition in post- conﬂict or post-colonial states, fostering the values of citizenship and social cohesion becomes a social justice imperative. Social cohesion, in particular, addresses critical development challenges where collective action is required to (re) build societies and regain trust in authorities. This is particularly important in conﬂict or post-conﬂict states, where values of citizenship and activities that foster social cohesion are under threat from being realized. South Africa, currently in its third decade of democratic rule, has been plagued with high levels of inequalities to such an extent that it has been listed one of the most unequal countries in the world. Coupled with high levels of crime and violence, poor leadership, infrastructural challenges, such as water and electricity outages, corruption, high levels of unemployment, and distrust in authority, it is obvious that the country needs a socially cohesive society. Similarly, in Zimbabwe, Gavin (2022, Para. 1) notes that “conditions for the people of Zimbabwe continue to go from bad to worse”. Politically, the electoral process has been critiqued for inconsistencies and a lack of credibility given the outcomes over the past several decades. Citizenship and social cohesion as foundational to democracy With the merging of the military and the ruling party, Zimbabwe has often been referred to as a “military dictatorship” (Grignon, 2008). Sigauke (2019, 246) also mentions that Zimbabwe has “been characterized by hyperinﬂation, social hemorrhage, and political conﬂict . . . [and for] most of the 1990s and beyond, Zimbabwe has been characterized by a gradual economic decline characterized by rising unemployment, underdevelopment, and disillusionment with elite corruption.” The state of CPD in the Southern African Development Community (SADC) region This section provides an overview of CPD developments in the SADC region as a way of providing a situational analysis for countries within this consortium. The professional development of teachers has piqued the interest of governments and researchers alike as they acknowledge the positive eﬀect CPD has on learner performance and teacher and teaching quality. As such, multilateral organizations, such as UNESCO, have engaged heavily with African states as a mechanism for improving the provision of public education on the continent. The UNESCO Regional Oﬃce for Southern Africa FIGURE 1 The interrelation and interdependence between citizenship, social cohesion and democracy. FIGURE 1 The interrelation and interdependence between citizenship, social cohesion and democracy. 03 Frontiers in Education frontiersin.org 10.3389/feduc.2024.1326437 Singh and Mukeredzi FIGURE 2 Core functions supported by the SADC Regional Framework on Continuing Professional Development (CPD) for Teachers (UNESCO, 2020). (ROSA) has, since 2015, initiated a series of meetings, workshops and consultations with SADC countries to generate evidence about the importance of improving the quality of teachers. From these initial discussions, two key priorities emerged−teacher standards and competencies, and CPD (UNESCO, 2020). In the context of CPD, the SADC countries, represented by senior government oﬃcials in charge of teachers, agreed that: • “countries needed to systematize and harmonize their teacher training policies and practices on CPD”; • “research was needed to collect more information and have a more in-depth understanding of the status of CPD practices for teachers in SADC countries”; and • “a SADC regional framework would help countries develop and/or strengthen their own national CPD programmes”, which includes a focus on TVET education (UNESCO, 2020, 5). In 2019 and 2020, UNESCO commissioned an investigation into the state of CPD in the SADC region (SADC and UNESCO, 2019). The study was focused on Botswana, Eswatini, Lesotho, Malawi, Mozambique, Namibia, South Africa, Zambia and Zimbabwe, where each presented a country case study that would inform the CPD Framework to improve CPD in the region. A synthesis of the country reviews, drawn from the draft SADC Regional Framework on Continuing Professional Development (CPD) for Teachers, is described below: FIGURE 2 Core functions supported by the SADC Regional Framework on Continuing Professional Development (CPD) for Teachers (UNESCO, 2020). Continuous professional Continuous professional development in South Africa and Zimbabwe: policy frameworks and learning opportunities to promote citizenship and social cohesion in schools Frontiers in Education frontiersin.org The state of CPD in the Southern African Development Community (SADC) region The review of CPD for teachers in the SADC region illuminates essential gaps in the professional development of teachers, which is a crucial indicator of the state of teacher support for professional development in these countries. Although there have been numerous developments instituted to improve the professional development of teachers who are currently in the classroom, these eﬀorts are often fragmented and occur predominantly in aﬄuent pockets, which highlights the levels of inequality within and between these education systems. “All countries have documents that declare and acknowledge the importance of CPD in the region, as a way of improving the quality of education and working toward realizing SDG4. However, not all documents are presented as policies. A CPD policy overview in the region ranges from clear stand-alone CPD policies, to draft policies, or no policies. Where CPD policies are available, the governance structures diﬀer ranging from autonomous institutions to Ministries of Education, with the overall responsibilities for implementing CPD in most countries residing with the Ministry or Department of Education. In some instances, authority is also given to quasi-governmental organizations, teacher training institutes and local government departments. At this stage, the synthesis could not establish the eﬃcacy of centralized versus decentralized models and it was highlighted that more research needed to be commissioned to explore these diﬀerences. Whilst there is clear evidence in most states of training to improve teacher knowledge and skills, irregular and unpredictable funding remains a challenge, as only a few states have dedicated CPD budgets. Many states in the region depend on donor or external funding, and this has implications for how CPD is deﬁned and what is prioritized. With the exception of Mozambique and Namibia, no reference was made to TVET sector or intersectional issues. Overall, the review found that CPD in the region is either [sic] inadequate, ineﬀective, not available or completely absent. There was also no evidence of rigorous monitoring and evaluation of CPD” Adapted from the Draft SADC Regional Framework on Continuing Professional Development (CPD) for Teachers. Zimbabwe provide an oversight of the workload of teachers (Department of Basic Education [DBE], 2003), the roles, competencies and standards for teachers (Department of Education [DoE], 2000), evaluation of teacher performance (Department of Basic Education [DBE], 2013), and the teachers’ conditions of service (South Africa [SA], 1998), amongst others. provide an oversight of the workload of teachers (Department of Basic Education [DBE], 2003), the roles, competencies and standards for teachers (Department of Education [DoE], 2000), evaluation of teacher performance (Department of Basic Education [DBE], 2013), and the teachers’ conditions of service (South Africa [SA], 1998), amongst others. In 2019, the Ministry of Primary and Secondary Education initiated discussions with various educational stakeholders, including teachers, about the need to develop a CPD Framework in the country. The intended purpose of this framework, as noted by the Ministry, is to “. . . guide the design and implementation of continuing professional development programmes for the teachers and learners . . . [and] to improve the quality of teaching and learning practices, and raise student learning outcomes at all levels of the education system . . . to contribute toward the attainment of the Sustainable Development Goal Four (SDG4)” (Ministry of Primary and Secondary Education, 2019, Para. 2). The need for a CPD framework also emerged from the newly instituted curriculum, launched in 2017, that now required teachers to “acquire a whole set of new competencies based on the principles” of this new curriculum (Ministry of Primary and Secondary Education, 2019, Para. 4). With regards to citizenship, social cohesion, and democracy, several policies are speciﬁcally geared toward realizing this. Firstly, the Manifesto on Values, Education and Democracy (Department of Education [DoE], 2001), which directly addresses the need to unite citizens through processes of social cohesion after many years of separate development in the country. The National Policy Framework for Teacher Education (Department of Education [DoE], 2006) employs schools to impart the necessary, skills, knowledge and dispositions to learners that promote the values of citizenship, social cohesion and democracy. More recently, the Department of Arts and Culture (2012) released a National Strategy for Developing an Inclusive and Cohesive Society and the Department of Justice (2016) released a National Action Plan to combat racism, racial discrimination, xenophobia and related intolerance. Both these policies advocate strongly for the values of citizenship and social cohesion to be upheld. Zimbabwe Citizenship, social cohesion, democracy and social justice are transversal themes in most of the country’s policies. Thus, the realizing of citizenship and social cohesion is seen as a systemwide challenge, with various departments of government advocating for this, not making it the sole responsibility of the Department(s) of Education, through the institutions and the work of teachers. The Ministry notes that it has put in place a number of eﬀective in-service training programmes to capacitate teachers to teach the new curriculum. However, these programmes tend to be ad hoc with an overemphasis on certain subjects and an underemphasis on others. Thus, the new framework will be able to identify gaps in the development of teachers and supervisors, which should culminate in better and more impactful CPD opportunities for teachers (Ministry of Primary and Secondary Education, 2019). Regarding the new curriculum, between Early Childhood Education and Form 6, there is evidence of several learning areas that tend to focus on citizenship, nationhood, heritage and social cohesion. Table 1 below provides an overview of these curriculum oﬀerings: From a curriculum perspective, citizenship and social cohesion are predominantly housed in a subject called “Life Orientation” or “Life Skills.” Although this subject has aspects of citizenship and social cohesion as part of its content, these values and concepts also arise in history and languages and also serve as carrier subjects. CPD for citizenship and social cohesion for teachers in the country does not meet the enthusiasm of the policy landscape. Recent training and interventions in CPD for citizenship and social cohesion include the Institute for Justice and Reconciliation’s Teaching Respect for All programme, a UNESCO initiated endeavor. Similarly, the Cape Town Holocaust Center runs a programme called “The Holocaust: Lessons for Humanity,” which emphasizes the importance of tolerance and mutual respect. The National Professional Teachers Organization of South Africa (NAPTOSA), a teacher union, also provides programmes to schools, but this is often at a fee, thus only for schools that can aﬀord to pay. Their programme oﬀering also provides training on teaching and learning in diverse contexts and how to achieve quality education with a values approach. Shikaya is another organization that oﬀers various programmes all aimed at citizenship, social cohesion and social justice. South Africa South Africa has an impressive policy landscape dedicated to the professional development of teachers. This is due to the prevailing consensus that teachers are central to transformation in a post-apartheid context. Policy frameworks in the country distinguish between the professional development of teachers and how the profession is governed. Initial teacher education is governed by the Minimum Requirements for Teacher Education Qualiﬁcations (MRTEQ) and professional development for qualiﬁed teachers is governed by the Integrated Strategic Planning Framework for Teacher Education and Development (ISPFTED). In terms of teacher governance, there are several frameworks that The framework is and continues to be developed in consultation with departments of education, teaching unions, teaching councils, civil society organizations, and the private sector. The framework aims to support the following functions (Figure 2). 04 frontiersin.org Singh and Mukeredzi 10.3389/feduc.2024.1326437 10.3389/feduc.2024.1326437 Frontiers in Education Study A: South African teachers’ understanding and practices of citizenship This sentiment is echoed by Sigauke (2011), whose study notes that individuals rarely discuss political issues because there is suspicion that talking about politics may result in victimization. This means that, although there are policies available or currently being developed that advocate for citizenship and social cohesion, the practice of this remains a challenge. This has two implications. Firstly, it will impact how aspects of citizenship and social cohesion are taught and, secondly, it will also impact how teachers are trained to teach it. With widespread fear of speaking out against the government, teachers limit the content of their discussions about citizenship and social cohesion in the classroom which hampers the development of what Westheimer and Kahne (2004) refer to as “authentic citizens.” The authentic citizen can move beyond a passive understanding of citizenship toward a more justice- orientated position in which citizens can analyze and address social injustices. The political turmoil that occurred after the 2000s has resulted in the erosion of democracy and an increase of populist rhetoric from the ruling party justifying its position, which has silenced the public from openly voicing their views against the economic challenges and corruption in the country (Sigauke, 2019). Further to this, the current political regime disallows teachers from fulﬁlling their role as transforming agents by pursuing social justice. The current civics curriculum does not contain any controversial topics to ensure that teachers do not contradict the views of the state. Attempts to make human rights a standalone subject “failed due to the same reason that teachers are hesitant to teach issues they regard as politically sensitive that would get them in trouble with the ruling party” (Sigauke, 2011). The narrow view of nation building, citizenship and social cohesion is also reﬂected in teacher professional development, both in initial teacher education and in CPD programmes and is thus inconsistent with the true values of citizenship and social cohesion. Matereke (2012, 97) notes that this political rule renders “both the school system and teachers as mere functionaries of the status quo, thus constricting the public sphere and eroding civil liberties, these being the very elements which enable citizens to fully participate in the political process and to hold public oﬃcials and institutions accountable. Study A: South African teachers’ understanding and practices of citizenship It is these developments that bring the dual crisis of citizenship and education into purview.” The data for the ﬁrst study on citizenship were part of a larger study that investigated learners’ and teachers’ understanding and experiences of citizenship in four high schools in South Africa (Singh, 2020). These four public high schools were stratiﬁed by quintile (poverty index) and geography. A total of eleven teachers participated in this study. The sample was purposive as it selected teachers who taught English, Life Orientation or History as subjects that are often noted as being carrier subjects for concepts such as citizenship and social cohesion and are often part of the curriculum. This study did not ascertain any views relating to teachers’ understanding of social cohesion as the study was citizenship-focused; as such, the ﬁndings presented will only reﬂect their views, practices, and CPD experiences relating to citizenship. Teachers participated in face-to-face semi-structured interviews. Each interview was recorded and then transcribed using professional transcribers. Respondents had the option of having the interview done in English, Afrikaans or isiXhosa as these were the dominant languages in the Western Cape Province. Some guiding questions included: 1. What is their understanding of citizenship and how is this depicted in practice? 2. What kind of professional development activities do teachers at the school participate in? 3. Has there been speciﬁc training on citizenship, social cohesion, or similar? Elaborate? Table 2 below provides an overview of the respondents in Study Zimbabwe Their programmes include “Facing History, Facing Ourselves” in collaboration with the USA programme of the same name; they also run the Creating Inclusive and Caring Schools Programmes, which has similar objectives. Whilst there may well be several other oﬀerings, often at school or district level, few opportunities exist for teachers in the country and, in many cases, the CPD is delivered by an NGO which often requires payment, making access a challenge for most schools. In summary, CPD for citizenship and social cohesion in the country is fragmented, inconsistent with no cemented national oﬀering. The recently revised Zimbabwe Education Amendment Act (2020) has also focused attention on contextual and aﬀective challenges. For example, the Act prohibits pregnant girls from being expelled; requires schools to provide menstrual health facilities to learners; ensures that parents do not deprive their children from receiving an education; forbids corporal punishment; upholds respect for learners’ human dignity; and makes provision for schools to have infrastructure to support disabled learners. Despite all these progressive developments, there is no mention of inclusive and equitable education as a priority, despite this being a cornerstone of the SDG4. These frameworks and amendments emphasize the state’s dedication to providing quality education and promoting the ideals of democracy−including citizenship and social cohesion. However, Hapanyengwi-Chemhuru and Shizha (2011, 108) caution that “citizenship attainment cannot be separated from the political TABLE 1 Overview of speciﬁc carrier subjects for citizenship and social cohesion in the Zimbabwean Curriculum. Phase Subject/s 1 ECD−Grade 2 Family and Heritage Studies 2 Grade 3–7 Family, Religion and Moral Education Heritage and Social Studies 3 Forms 1–4 Family and Religious Studies Heritage Studies 4 Forms 5–6 None TABLE 1 Overview of speciﬁc carrier subjects for citizenship and social cohesion in the Zimbabwean Curriculum. Phase Subject/s 1 ECD−Grade 2 Family and Heritage Studies 2 Grade 3–7 Family, Religion and Moral Education Heritage and Social Studies 3 Forms 1–4 Family and Religious Studies Heritage Studies 4 Forms 5–6 None TABLE 1 Overview of speciﬁc carrier subjects for citizenship and social cohesion in the Zimbabwean Curriculum. TABLE 1 Overview of speciﬁc carrier subjects for citizenship and social cohesion in the Zimbabwean Curriculum. Frontiers in Education 05 frontiersin.org frontiersin.org Singh and Mukeredzi 10.3389/feduc.2024.1326437 to passive citizenship having catastrophic social, economic and political consequences. to passive citizenship having catastrophic social, economic and political consequences. ideology of the state”. Methodology “The content knowledge required to teach pupils about alternative forms of government, democracy, freedoms and human rights and to expand their knowledge of politics beyond the politics of coercion introduced by ZANU PF is lacking in schools. While teachers might be aware of other alternative forms of government and ideologies, they are afraid of informative and critical pedagogy that frees learners from developing a narrow tunnel vision of the political and governance system in Zimbabwe. Teachers who might want to develop rational and critical thinking and analytical skills are afraid of being labeled “enemies of the state” (Hapanyengwi- Chemhuru and Shizha, 2011, 117). This section discusses the methodologies used in procuring the data and the analysis process. It also provides an overview of each of the studies presented in this paper. Frontiers in Education Zimbabwe The authors further note that the repressive nature of the current regime has led to many citizens, including teachers, avoiding engaging in policy issues and challenging the state due to fear of repercussions as noted in the extract below: Table 2 below provides an overview of the respondents in Study A. Study B: Zimbabwean teachers’ understanding and practices of citizenship and social cohesion The Zimbabwean case study was carried out in four schools across three provinces−Harare, Midlands and Masvingo. Two schools were urban, one was rural (50 km) and the fourth was in a peri-urban (11 km) setting. This was a qualitative case study where data were generated through an open-ended questionnaire. This study sought to understand teachers’ understanding, their practices, and CPD experiences related to citizenship and social cohesion. The overview presented here suggests that Zimbabwe’s education system, through its curriculum and teacher pedagogies, conveys a distilled version of citizenship and social cohesion to learners which can threaten a deliberative democracy and lead 06 frontiersin.org 10.3389/feduc.2024.1326437 Singh and Mukeredzi TABLE 2 Overview of respondents in Study A. Respondent School Type Gender Quintile Geography 1 Teacher A (Lisa) High School Female 1 Rural 2 Teacher B (Raymond) High School Male 1 Rural 3 Teacher C (Zimkitha) High School Female 1 Urban 4 Teacher D (Tebalelo) High School Male 1 Urban 5 Teacher E (Jonathan) High School Male 5 Rural 6 Teacher F (Gillian) High School Female 5 Rural 7 Teacher G (Lilith) High School Female 5 Rural 8 Teacher H (Letitia) High School Female 5 Urban 9 Teacher I (Saleem) High School Male 5 Urban 10 Teacher J (Sylvia) High School Female 5 Urban 11 Teacher K (Reginald) High School Male 5 Urban it is often used as a safeguard in education research to ensure the research is valid and credible. At least three teachers at each school were interviewed, which helped conﬁrm the school context, availability of CPD, and what teachers do in the classroom. Participants were selected through snowball sampling. One teacher was identiﬁed at each school and asked to distribute the open-ended questionnaire to their colleagues in the History department who were teaching National Social Security Studies (NASS). Seventeen secondary school teachers participated in the study. Some guiding questions for the study included: Participants were selected through snowball sampling. One teacher was identiﬁed at each school and asked to distribute the open-ended questionnaire to their colleagues in the History department who were teaching National Social Security Studies (NASS). Seventeen secondary school teachers participated in the study. Some guiding questions for the study included: The data for the questionnaire administered in Study B was also validated using triangulation. Frontiers in Education Table 3 below provides an overview of the sample for Study B: Table 3 below provides an overview of the sample for Study B: Both studies received ethical clearances through the Cape Peninsula University of Technology Ethics Board and the South African study also received ethics through the Western Cape Education Department. At all times, the research protocol was observed, and respondents participated voluntarily. They were also informed that they could withdraw their participation at any time without fear of repercussions. The data were analyzed using content analysis (see Patton, 2014) as this was the most eﬀective way to ascertain keywords, themes, and concepts from qualitative data. Within the domain of content analysis, the authors selected a conceptual analysis approach as the main goal was to investigate occurrences of phrases and terms as well as incidences (Krippendorﬀ, 2018). Further to this, content analysis was used because “qualitative content analysis is not linked to any particular science, and there are fewer rules to follow . . . therefore, the risk of confusion in matters concerning philosophical concepts and discussions is reduced” (Bengtsson, 2016, 8). This analytic technique was paired with Braun and Clarke’s (2006) iterative thematic analysis process for thoroughness. The authors favored qualitative techniques for this research because it “is uniquely positioned to provide researchers with process- based, narrated, storied data that is more closely related to the human experience” (Stahl and King, 2020, 26). Credibility refers to how “true” the ﬁndings are. In this research, teachers’ views from various schools and geographical spaces were triangulated to establish credibility. Transferability refers to the extent to which the study could be adapted for another context. Whilst qualitative research does not necessarily allow for replicability, patterns, descriptions, and experiences may be observed if the study was conducted in another context, and the ﬁndings would highly likely align. The ﬁndings of Study A and Study B align with other studies relating to teachers’ experiences of CPD and citizenship and social cohesion, such as Raanhuis (2022) and Sayed et al. (2018). Dependability refers to the accuracy of the interpretation of the ﬁndings. In this research, each author looked at and interpreted the ﬁndings, and the analysis was compared to ensure there was no intended bias in the reporting process and that the experiences and responses of participants were accurately represented. frontiersin.org Study B: Zimbabwean teachers’ understanding and practices of citizenship and social cohesion Teachers from various geographical contexts (urban, peri-urban, and rural) were included in the study, and all their responses, as it relates to the focus of this paper, were included in the analysis and presented in this paper. As such, all voices were included, and any outliers or inconsistencies would have been highlighted and discussed. Further to this, the questionnaires only used open-ended questions, which means respondents were not limited in how they could respond, which is often a limitation of closed-ended questionnaires. 1. What is your understanding of citizenship and social cohesion? 1. What is your understanding of citizenship and social cohesion? 2. Have you received any training or staﬀdevelopment on citizenship and social cohesion? 3. How do you implement the values of citizenship and social cohesion in your classroom? A key diﬀerence in validation techniques between qualitative and quantitative is where the responsibility of the validation lies. In quantitative research, much emphasis is placed on having “faultless” instruments. In the context of qualitative research, the responsibility for validation and credibility lies with the researcher. Here, the work of Guba and Lincoln (1985) remains a useful checklist for establishing the trustworthiness of data. These authors contend for a research study to be evaluated as trustworthy; it needs to establish credibility, transferability, dependability, and conﬁrmability (Guba and Lincoln, 1985). Table 3 below provides an overview of the sample for Study B: Lastly, Guba and Lincoln (1985) speak The data for the interviews conducted in Study A was validated through the process of triangulation, as Brown (2018) laments 07 frontiersin.org 10.3389/feduc.2024.1326437 Singh and Mukeredzi Respondent School Type Gender School Marker Geography 1 Teacher A (Arthur) High School Male BVD Urban 2 Teacher B (Colin) High School Male BVD Urban 3 Teacher C (Jeﬀrey) High School Male BVD Urban 4 Teacher D (Caitlin) High School Female BVD Urban 5 Teacher E (Katy) High School Female BVD Urban 6 Teacher F (Kwazi) High School Male MKO Urban 7 Teacher G (Jason) High School Male MKO Urban 8 Teacher H (Jake) High School Male MKO Urban 9 Teacher I (Solomon) High School Male MKO Urban 10 Teacher J (Adam) High School Male MSV Peri Urban 11 Teacher K (Kenneth) High School Male MSV Peri Urban 12 Teacher L (Cody) High School Male MSV Peri Urban 13 Teacher M (Liza) High School Female MSV Peri Urban 14 Teacher N (Joseph) High School Male BNFL Rural 15 Teacher O (Oscar) High School Male BNFL Rural 16 Teacher P (Laura) High School Female BNFL Rural 17 Teacher Q (Grace) High School Female BNFL Rural ascertain what teachers understood citizenship and social cohesion to mean and what values they associated with these terms. The ﬁndings that respond to the ﬁrst research question are depicted in the two tables below. Table 4 depicts the responses for Study A (South Africa) and Table 5 depicts the responses for Study B (Zimbabwe). about conﬁrmability, which refers to the degree to which the ﬁndings are shaped by the participant’s responses and not the researcher’s own bias and personal agendas. In this research, the authors ensured to follow a sound analysis of the data by using a double approach of content analysis and iterative thematic analysis. The triangulation of the ﬁndings also supports the neutrality in how the data was represented. The ﬁndings for teachers in South Africa suggest that citizenship is predominantly about belonging, as noted by Lisa, Raymond, Zimkitha, Sylvia and Gillian; about rights, as noted by Reginald; and about responsibilities, as noted by Tebalelo, Lilith and Letitia. The value of respect for others was also a recurring theme amongst participants. Sylvia’s comment suggests that her understanding of citizenship involves a social contract and a mutually beneﬁcial relationship between the citizen and the state. Findings This section discusses the ﬁndings that responded to the following research questions: (1) What are teachers in South Africa and Zimbabwe’s understanding of citizenship and social cohesion? (1) What are teachers in South Africa and Zimbabwe’s understanding of citizenship and social cohesion? (2) Have teachers in South Africa and Zimbabwe been exposed to CPD for citizenship and social cohesion? (3) How do teachers in Zimbabwe and South Africa incorporate the values of citizenship and social cohesion in their classroom? Table 3 below provides an overview of the sample for Study B: The responses from Jonathan and Saleem request that one should “forget the politics, forget who is running the country” and to enjoy the privileges of the country “without the political things attached”. It is quite concerning for educators to hold these views because political participation is key to realizing full citizenship (see Marshall, 1950; Veldhuis, 1997). For Marshall (1950), citizenship, as a right, includes civil, social and political rights, the latter which require citizen engagement, not disengagement, as noted by Saleem and Jonathan. Similarly, Veldhuis (1997) notes that citizenship has four dimensions including the social, the cultural, the economic and the political with each needing to be exercised equally for citizenship to be fully realized. Whilst Jonathan and Saleem seemingly dismiss any discussion or engagement with the political element of citizenship, this could be due to the possible negative repercussions and backlash that may occur if they did engage in these discussions. This omission is of concern as schools are learning and socializing institutions and what learners learn (or don’t learn) can shape and cement the ideas and values they hold as adults. Therefore, we reiterate the importance of fostering the Frontiers in Education frontiersin.org Teachers’ understanding of citizenship and social cohesion Investigating teachers’ views and understanding of citizenship and social cohesion is important because it has implications for their teaching practices. Pajares (1992) notes that the beliefs and views teachers hold inﬂuence their judgment and directly impact their behavior in the classroom. Hannula (2004) maintains that the aﬀective dimensions that impact the quality of teaching and learning include beliefs, emotions, attitudes, values, ethics and morals. The studies on which this data was procured aimed to 08 frontiersin.org Singh and Mukeredzi 10.3389/feduc.2024.1326437 TABLE 4 Study A: Teachers’ understanding of citizenship (South Africa). TABLE 4 Study A: Teachers’ understanding of citizenship (South Africa). Respondent Understanding of Citizenship Understanding of Social Cohesion 1 Teacher A (Lisa) I am born in South Africa, so the fact that I am born here makes me a citizen and to be a good citizen you have to respect peoples and look after the elderly. . . and to uphold what our parents and grandparents have taught us. . . n/a 2 Teacher B (Raymond) . . . citizenship means that I am born here and that I accept the values of the constitution and I try to live and strive towards living the constitution as a responsible citizen . . . n/a 3 Teacher C (Zimkitha) . . .you have to know who you are, in which country you belong to, are you a South African?, a Nigerian? and if you are a particular citizen of a country, you must know your rights and what to do. And being a citizen or with citizenship who has accountability and responsibility. n/a 4 Teacher D (Tebalelo) I think it simply means that is those people that abide by the law, or they abide by the law in terms of paying the taxes, in terms of respecting other people, in terms of like also respecting their environment, taking care of and of being responsible for your actions – n/a 5 Teacher E (Jonathan) Its to become a decent South African. To serve your country, and forget about politics, forget who is running the country. What I miss, if I compare South Africa at the moment to say...I don’t like the English, Britain, but compare their culture with our culture, we’re missing something at the moment. . . n/a 6 Teacher F (Gillian) If you live in a city or town. . ., then I’m part of [that city /town]. Teachers’ understanding of citizenship and social cohesion And that means that I belong to [that city/town] and I’m part of [that city or town], so it’s very important that I then be part of the community and do everything which is part of the community and ﬁt in and I belong to [that city/town] I have to keep [that city/town] name up and that then inﬂuences my social life, it inﬂuences my school, and then as a teacher, I also have to be part of [that city/town] and do it in a good way. . . .have a good name, and so that the children and the parents can respect me. n/a 7 Teacher G (Lilith) someone who obeys the law, who wants to create a decent environment n/a 8 Teacher H (Letitia) It can be lots of things, but essentially if it be your responsibility if you want, if you contribute to part of a greater system. . .. and accountability . . . because you can’t expect to be considered part of a whole if you’re not conforming to what is expected of that situation. n/a 9 Teacher I (Saleem) . . .to me its having the beauty of living in the country and enjoying the privileges of that country without the other political things attached. n/a 10 Teacher J (Sylvia) It can be very broad. . . . I would think not just belonging, but also loyalty and vice versa . . . like the citizens be there for the state, the state must be there to cater to maybe all their needs. . . n/a 11 Teacher K (Reginald) Knowing your rights, knowing our constitution, being aware of issues that are of discrimination, knowing your limitations, understanding people, being involved in outreach, making sure that other people share your knowledge and train them to get them, because, so constantly there’s a cycle and you need to be part of that cycle. You need to be part of everything is a cycle in this universe, and you need to be part of the cycle, whether it be resources, be aware of, whether it be the food you eat, your ﬁtness,.. and you’re not an island, and therefore anybody who is diﬀerent you have to engage, you have to make sure you build a bridge with that person. . . Frontiers in Education Frontiers in Education Teachers’ understanding of citizenship and social cohesion n/a values of citizenship and social cohesion in schools, with CPD for teachers being one of these mechanisms to support this process. be that their understanding of the concept may be more tangential and diverse rather than them dismissing the political dimension as important. Alternatively, teachers disengage due to the potential negative backlash. For social cohesion, all respondents noted relationships, connectedness, interaction and integration. Working toward a national consciousness and nation building (Oscar), anti- individualistic behavior (Lisa) and solidarity (Solomon, Adam) also emerged as viewpoints. The ﬁndings for teachers in Zimbabwe suggest that citizenship is fundamentally about belonging as noted by Caitlin, Katy, Jason, Jake, Adam, Kenneth and Oscar. Others noted that it is about roles and duties of the citizen (Kwazi, Arthur, Solomon, Cody, Joseph, Laura and Grace) as well as a relationship with the state (Grace), civil rights (Kwazi), identity and patriotism (Kenneth), inclusion (Jake) and enabling choice and decision making abilities (Lisa). Although none of the respondents overtly mentioned political engagement as key to their understanding of citizenship, it could In comparison, in both studies, the manner in which citizenship and social cohesion is understood classiﬁes these concepts as an on-going, daily, interactive process (such as respecting others, 09 frontiersin.org Singh and Mukeredzi 10.3389/feduc.2024.1326437 TABLE 5 Teachers’ understanding of citizenship and social cohesion (Zimbabwe). Respondent Understanding of Citizenship Understanding of Social Cohesion 1 Teacher A (Arthur) The privileges of someone. . .acknowledging and promoting roles and duties. . . The link between people in a society 2 Teacher B (Colin) Legally belonging to a given country The act of individuals and groups in society working together in harmony towards a shared goal 3 Teacher C (Jeﬀrey) Means a position of being a citizen of a particular country Refers to the strength of relationships, connectedness and the sense of solidarity 4 Teacher D (Caitlin) State of belonging to a nation state by birth, origin or descendant or registration as per the legal given provisions of a particular state or country. . . Teachers’ understanding of citizenship and social cohesion Co-operation / unity in the various interactions among people of a group inhabiting a speciﬁc geographical area 5 Teacher E (Katy) When one belongs to a given state as enshrined in the constitution – could be by birth or as stated in the constitution The ability to live and work together in a given social setting e.g., community 6 Teacher F (Kwazi) Civic issues, rights and responsibilities Unity within groups at diﬀerent levels e.g., family, community and country 7 Teacher G (Jason) Sense of belonging to a state or country Unity within and across groups in a nation or country 8 Teacher H (Jake) Position or status of being a citizen of a country, ﬁghting discrimination, social exclusion and inequalities by to building social capital, Living according to good citizenship values Strength of relationships and the sense of solidarity among members of a community 9 Teacher I (Solomon) Social rights, obligations and engaging in positive behaviour Shared values, a sense of solidarity between members of a community 10 Teacher J (Adam) Status of being a citizen of a particular country. Unity, strength of a relationship, a sense of solidarity between members of a society 11 Teacher K (Kenneth) Identity, patriotism, ownership, belonging to a nation, sovereignty, desire to develop one’s nation When people work together for a common good, unity, team work and interaction of members in the society. 12 Teacher L (Cody) The social responsibilities and accountabilities expected of persons living within set boundaries The social bonds within communities that enhance peace, social and economic development 13 Teacher M (Liza) It’s about enabling people to make their own decision and also be able to take responsibility for their own lives and their communities It’s about people coming together, being interactive, cooperative and collaborative in every aspect of their social life. Its communally based and its not individualistic 14 Teacher N (Joseph) Acquisition of civic responsibilities, and values for the for the formation of good citizens. Teachers’ understanding of citizenship and social cohesion Integration of members of a society for nationhood 15 Teacher O (Oscar) Members of a common society – cherishing common norms and values in a patriotic way Unity or oneness for achieving a common goal which is a national consciousness and nation building 16 Teacher P (Laura) The status of being a citizen along with the rights, duties, and privileges of being a citizen Unity of a purpose amongst members of a community 17 Teacher Q (Grace) The relationship between an individual and the state where one understands and fulﬁlls their rights and duties within their society The degree of social integration and inclusion in communities Booysen’s (2015) study is also illustrative of South African youth’s views of political participation. Booysen’s study included focus groups with youth in three diﬀerent provinces in South Africa about their views on political participation and revealed that learners will only vote if they “get” something in return, such as jobs. In the absence of this, they do not see the necessity. Similarly, in the study conducted by Singh (2020) learners noted that “. . .distrust of leadership, corruption, high levels of unemployment, poverty, and apathy as reasons why they chose to not participate politically” (p, 222–223). This was also evident in the results of the 2019 national elections, where the youth turnout was very low. Similarly, in Zimbabwe, where the 2012 National Population Census revealed that 76.1% of the population are under the age of 34 and about 68% of this group are between the ages of 15 and 34. Although being the largest demographic segment, their civic participation is very low (Future Africa helping others, helping the elderly and the community, and being inclusive, etc.) but also as a goal to be achieved (solidarity, peace, and economic development). For both the South African and Zimbabwean teachers, the political foundations of citizenship and social cohesion are weak. This means that teachers either overtly disengage from politics, as in the case of South Africa, or subtly disengage by not mentioning this in their understanding of these concepts, as in the case of Zimbabwe. Singh et al. (2018) argue that it is important to understand the views that teachers hold because, once known, interventions can be developed to improve practice. If teachers are disengaging from politics and transferring these disengaged views onto learners, this may lead to catastrophic consequences for enabling a democracy. frontiersin.org Frontiers in Education Teachers’ exposure to CPD for citizenship and social cohesion and associated classroom practices This section responds to the research questions 2 and 3 which are: “Have teachers in South Africa and Zimbabwe been exposed to TABLE 6 Teachers’ exposure to CPD for citizenship and their self-reported associated classroom practices (Study A). Respondent Received CPD for citizenship/ social cohesion Do you practice/ implement the values of citizenship and social cohesion in your classroom? 1 Teacher A (Lisa) No, not at all It’s quite really diﬃcult,. . . if I have to look at Grade 8 and Grade 9, many of them when we talk about sex, about gays and all this stuﬀ, lesbians and all that stuﬀ, then there are a few of them who are frivolous and laugh about these topics. But for me, and as you say, it’s very sensitive,.. there are homosexual kids in the class. . .but I say, we must learn to respect these people because these people have rights. So I’ll always say they have their rights, we can’t discriminate against them or whatever, especially if the kid is in the class, and you know 2 Teacher B (Raymond) No [referring to the practices of teachers at the school] No, not at all. Because we, many of our teachers in our time were at a university based on a certain race. So many of us at East Cape, or at the Colleges or at the Tech. The new type of teacher being trained is now being trained much more in a multi-racial culture. . . 3 Teacher C (Zimkitha) Yes when the child enters our school, then that child learns discipline in terms of being responsible 4 Teacher D (Tebalelo) Yes if you are in class teaching that child, but if you’re seeing that child is doing something wrong, then it is your duty to speak with that child. . . 5 Teacher E (Jonathan) Yes If I can explain it to you. Once a guy comes into my oﬃce, let’s say he’s transgressing a rule, say for instance he stole something then my approach won’t be, you stole something. We try to say, what value did you not obey? Respect for other people’s property? So we throw this whole thing towards values. 6 Teacher F (Gillian) No I don’t know. . .,sometimes you feel that children get more diﬃcult and diﬃcult and always to cope with them isn’t so easy. Teachers’ understanding of citizenship and social cohesion Studies conducted in South Africa about the political engagement of youth found that, although youth are pro-democracy, their civic and political engagement is very low (Mattes and Richmond, 2014). 10 frontiersin.org 10.3389/feduc.2024.1326437 Singh and Mukeredzi Forum, 2023). The Zimbabwe Electoral Commission notes that, out of about 5.6 million registered voters, 44% constitute youth between the ages of 18 and 34. Booysen (2015) maintains that a reason for this could be due to youth social rights and basic needs that are not being met. Another explanation could be the socialization process at school where teachers do not readily engage learners about politics. This is substantiated by Willeck and Mendelberg (2021) who believe that formal education is fundamental to encouraging political participation. Several others also echo this sentiment, for example, Nie et al. (1996, 2) note that there is a consistent and overwhelming correlation between formal education and political participation of youth and that, in formal educational attainment, “the primary mechanism behind citizenship characteristics is basically uncontested”). Willeck and Mendelberg (2021, 89) mention that “the link between education and political engagement is among the most replicated and cited ﬁndings in political science” and Verba et al. (1995) state that, if scholars could use one variable to predict voting patterns, the most reliable and variable would be the level of education. Forum, 2023). The Zimbabwe Electoral Commission notes that, out of about 5.6 million registered voters, 44% constitute youth between the ages of 18 and 34. Booysen (2015) maintains that a reason for this could be due to youth social rights and basic needs that are not being met. Another explanation could be the socialization process at school where teachers do not readily engage learners about politics. This is substantiated by Willeck and Mendelberg (2021) who believe that formal education is fundamental to encouraging political participation. Several others also echo this sentiment, for example, Nie et al. (1996, 2) note that there is a consistent and overwhelming correlation between formal education and political participation of youth and that, in formal educational attainment, “the primary mechanism behind citizenship characteristics is basically uncontested”). Willeck and Mendelberg (2021, 89) mention that “the link between education and political engagement is among the most replicated and cited ﬁndings in political science” and Verba et al. Teachers’ understanding of citizenship and social cohesion (1995) state that, if scholars could use one variable to predict voting There is also an assumption that individuals who are teachers are all good and wholesome individuals who are passionate about advancing democracy. This is not the case as teachers have their own histories, life experiences, political, religious and cultural beliefs that may mitigate against the values of democracy. As such, to ensure learners are being socialized for a democratic society, teachers require focused professional development to guide this process. Teachers’ exposure to CPD for citizenship and social cohesion and associated classroom practices Because ja, how to discipline them and after so many years in teaching, I believe I can, but sometimes you just feel I can’t go on any longer. . . 7 Teacher G (Lilith) No I wouldn’t say like to an exceptionally high level. . .. I’m a Christian as well, but I have respect for the other, . . .. I would never put my personal opinion and say, I will say if you are Christian, then you would do this, but if you are Muslim, . . . Because I feel that they are that appropriate age to make their own choices. 8 Teacher H (Letitia) No Well. . .you can talk about anything, and you can discuss anything, and you can write letters about anything, and you can analyse people from diﬀerent angles and all of that kind of thing. 9 Teacher I (Saleem) No [some teachers] discipline the child by putting the child outside the classroom, which is against the policy of the Department. And I say to them, why not tell the child to come and sit in front of your class? Start with that, you know. Or punish the child after school. Some teachers punish the child during an interval for example, which is also not right, because the child needs that break between sessions. 10 Teacher J (Sylvia) No Well, I have a lot of patience, and so I like talking and making the child understand, you know where the child maybe went wrong and then come back to talk, like maybe have a one-on-one. 11 Teacher K (Reginald) No For me it’s orientating them for life, it’s making sure that they carry those lessons through with them for life. Do you practice/ implement the values of citizenship and social cohesion in your classroom? Do you practice/ implement the values of citizenship and social cohesion in your classroom? It’s quite really diﬃcult,. . . if I have to look at Grade 8 and Grade 9, many of them when we talk about sex, about gays and all this stuﬀ, lesbians and all that stuﬀ, then there are a few of them who are frivolous and laugh about these topics. But for me, and as you say, it’s very sensitive,.. there are homosexual kids in the class. . .but I say, we must learn to respect these people because these people have rights. So I’ll always say they have their rights, we can’t discriminate against them or whatever, especially if the kid is in the class, and you know Frontiers in Education 11 frontiersin.org Singh and Mukeredzi 10.3389/feduc.2024.1326437 Teacher’s exposure to CPD for citizenship and social cohesion and their self-reported associated classroom practices (Study B). TABLE 7 Teacher’s exposure to CPD for citizenship and social cohesion and their self-reported associated classroom practices (Study B). Respondent Received CPD for citizenship/ social cohesion Do you practice/ implement the values of citizenship and social cohesion in your classroom? 1 Teacher A (Arthur) No Yes. In every lecture, attempts are made to ensure that captures most of the diﬀerent views. 2 Teacher B (Colin) Yes Yes. Appreciating diversity in teaching and learning. . . 3 Teacher C (Jeﬀrey) Yes Yes, by utilizing collaborating approaches to learning such as group work, class discussions and debates. Exposing learners to the history of diﬀerent social groups in the Zimbabwean society in order for them to value social and cultural diversity. 4 Teacher D (Caitlin) No Yes. Encouraging the sharing of unique cultural values and practices. . . 5 Teacher E (Katy) No Yes. Always bringing in aspects of a citizen’s rights when teaching topics such as democracy and human rights 6 Teacher F (Kwazi) No Yes. In teaching social studies, one of the goals is citizenship education so I teach my students about their rights and responsibilities. 7 Teacher G (Jason) No Yes. Through lectures on national pride and patriotism. 8 Teacher H (Jake) Yes Yes, by developing characters to become more mindful of our responsibilities, rights and roles in society. 9 Teacher I (Solomon) No Yes, through assisting other students in need of say fees and food. Accompanying and paying what is known as chema to a bereaved student’s family. Do you practice/ implement the values of citizenship and social cohesion in your classroom? 10 Teacher J (Adam) Yes Yes. Classroom discussions, debates, voting, exercising of their rights, conservation of natural resources, participation in projects for the community. . . 11 Teacher K (Kenneth) No Yes, participation in national events, e.g., national cleaning up events, being involved in soil conservation and encouraging critical thinking. . .. 12 Teacher L (Cody) Yes Yes, group work, educational tours. . .. 13 Teacher M (Liza) No Yes. Students are allowed to work together and also make choices. They are responsible and accountable for their choices. 14 Teacher N (Joseph) No Yes, we sing the national anthem and raise the ﬂag. We debate and dramatize national issues of a civic and social nature, we carry out educational tours as a way of learning. . . 15 Teacher O (Oscar) No Yes, I teach the values of both. . . we practice these values at assemblies, we participate in national events e.g., Africa Day, Independence Day, We also execute excursions to areas of national signiﬁcance. . .also collaborative research and entrepreneurial projects. 16 Teacher P (Laura) No Yes, respect individual diﬀerences, volunteering, empowering students as leaders, encouraging children to be a good citizen. 17 Teacher Q (Grace) Yes Yes, I hold debates on issues to do with human rights and responsibilities, taking ﬁeld trips around the community. CPD for citizenship and social cohesion?” and “How do teachers in Zimbabwe and South Africa incorporate the values of citizenship and social cohesion in their classroom?” Tables 6, 7 respond to these questions from Study A (South Africa) and from Study B (Zimbabwe) respectively. practices are essential for socializing learners toward enacting the democratic values. Goren and Yemini (2017) suggest that the characteristics and the approach of the adults involved in this socialization can determine the degree to which youth participate in public discourses and imbibe the values of citizenship and social cohesion. As such, all teachers, regardless of the subject, need to be consistently trained to ensure their classroom practices and pedagogies are consistent with the values of citizenship and social cohesion as their own understandings and views of these concepts may be misaligned. Although CPD is only one way of challenging teachers’ beliefs, it becomes impactful if the CPD is consistent. frontiersin.org Frontiers in Education Do you practice/ implement the values of citizenship and social cohesion in your classroom? The ﬁndings from the South African teachers demonstrate that, out of the eleven teachers, only two teachers (from an urban, quintile one school) noted that they had received CPD that focused on citizenship. Most of the teachers had not received any form of CPD on these topics. However, when teachers were asked whether they teach in a manner that promotes the values and practices of citizenship, all the responses were positive and examples of these strategies were provided. For example, one teacher explained that fostering tolerance in her class is her way of enacting these values (Lisa), others noted that it is enacted through positive disciplining techniques (Saleem, Zimkitha, Tebalelo, Jonathan and Sylvia), and Reginald believed in creating a class environment that allows learners to develop life skills. These pedagogical and classroom The ﬁndings from Study B demonstrate that, of the 17 teachers, only six noted that they had received CPD focused on citizenship and social cohesion. In this case, all teachers responded positively when asked whether they implement the values of citizenship in their classrooms. The methods used included: considering multiple viewpoints (Arthur); using a collaborative teaching style; having debates and encouraging discussions (Jeﬀery, Adam, Lisa); national 12 Singh and Mukeredzi 10.3389/feduc.2024.1326437 pride and patriotism (Jason); and participating in national events and educational tours (Kenneth, Cody). questions. This limitation was overcome by ensuring the analysis was more nuanced, acknowledging that it is quite possible that a diﬀerently phrased question may yield a diﬀerent response. This shows that teachers are implementing their understanding of citizenship and social cohesion in the classroom, informed by their own beliefs and views and not necessarily formal training. This begs the question: Are these teachers’ enacting values that are consistent with the values of democracy or are they perpetuating behaviors that encourage silencing of voices? A study by Leek (2019) also suggests that teachers themselves recognize the importance of being trained in the eﬀective dimensions of teaching and learning to encourage youth to become more active citizens. Leek’s (2019, 181) study notes that “the teachers expressed their concern about the lack of training on a whole spectrum of civic participation, including classroom psychology [and noted that] the times are changing, and the youth changes over time too, so each generation has diﬀerent needs as far as teaching is concerned. Do you practice/ implement the values of citizenship and social cohesion in your classroom? [As] a result, the teachers need to update their teaching methods accordingly and knowledge of how to teach in general.” Nonetheless, the ﬁndings demonstrate that in these studies, both South African and Zimbabwean teachers’ understanding of citizenship promotes the ideals of the “Responsible Citizen” (see Westheimer and Kahne, 2004), who privilege the ideals of respect for each other and the environment. Teachers in both counties also ﬁnd belonging to be an important aspect. Teachers avoid discussing politics and, in some instances, note that one should “forget the politics and forget who is running the country,” demonstrating either their political disengagement with the state or fear of discussing politics due to the possible negative repercussions. For the teachers in Zimbabwe, social cohesion is about solidarity, togetherness, patriotism, integration, unity and cooperation. Most teachers in these studies had not received training on citizenship or social cohesion. However, all teachers declared that they teach in a way that is consistent with their views of citizenship and social cohesion. Both countries have evidence of policies that promote the values of citizenship and social cohesion, with South Africa’s landscape being the more sophisticated of the two, but the oﬀering for CPD does not meet policy objectives in practice. Reasons for the inconsistent and fragmented oﬀering of CPD for citizenship and social cohesion include ﬁnancial challenges, departmental capacity and political interference. For learners to become active citizens who utilize their agency and embody the values of citizenship and social cohesion, one or two lessons a week on values and society from the civics curriculum is not suﬃcient. Learners need these values to pervade every aspect of their schooling lives, facilitated by teachers who are professionally trained and equipped to do so. This section discussed the ﬁndings that respond to the three research questions as noted earlier in this paper. The next section concludes the paper by summarizing the two studies, noting the contribution and limitation of the study, synthesizing the main ﬁndings, and reiterating the importance of teachers receiving consistent, good quality CPD on aﬀective concepts such as citizenship and social cohesion in order to build and support a ﬂourishing democracy. Teachers need to receive focused CPD on understanding and enacting the values of citizenship and social cohesion in the classroom because schools, through teachers, exercise a particular inﬂuence on the development of young people’s democratic knowledge and political literacy skills (Kisby and Sloam, 2014). Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Conclusion The aim of this paper was to illuminate South African and Zimbabwean teachers understanding of citizenship and social cohesion, their experiences of CPD relating to these concepts as well as the ways in which they implement these concepts in their classrooms. This paper is a reﬂection on and a response to the dire political challenges and ineﬃciencies experienced in these nations, where democracy is being threatened and its values are eroding. Teachers remain beacons of hope in these challenging times as their agentic nature is catalytic in socializing youth about democracy. However, when teachers are silenced and threatened through political interference, limiting how and what they can and cannot discuss in the classroom, democracy is undermined and weakened. The ﬁndings draw from two separate studies. The ﬁndings from the South African case draw from a larger study investigating learners’ and teachers’ understandings and experiences of citizenship in South Africa, where the discussion of CPD was a subset (see Singh, 2020). This study only focused on the concept of citizenship, not social cohesion, and is presented in this paper as such. The Zimbabwean case draws on a study that investigated teachers’ understanding of citizenship and social cohesion, their experiences of CPD relating to this, and the ways in which they implement this in their classrooms. A critical limitation of the study is that in some instances, the instruments posed leading Do you practice/ implement the values of citizenship and social cohesion in your classroom? Learners in South Africa and Zimbabwe have the right to be taught the full meaning of citizenship and social cohesion and to understand the practices associated with this. By doing so, they will know when democracy and democratic values are not being upheld. Well-trained and suitably equipped teachers are a critical factor to realize this because quality teachers beget quality education systems. Raanhuis (2021, 44) reiterates this by saying that “teachers in all contexts should be supported as agents of change, through CPD”. Thus, professional development for teachers, particularly in post-conﬂict and post-colonial states, becomes an issue of social justice and is critical in creating deliberative democracies. Frontiers in Education References Allender, J., and Allender, D. (2006). “How did our early education determine who we are as teachers?,” in Proceedings of the Sixth International Conference on Self-Study of Teacher Education Practices, eds L. M. Fitzgerald, M. L. Heston, and D. L. Tidwell (Cedar Falls, IA: University of Northern Iowa), 14–17. Allender, J., and Allender, D. (2006). “How did our early education determine who we are as teachers?,” in Proceedings of the Sixth International Conference on Self-Study of Teacher Education Practices, eds L. M. Fitzgerald, M. L. Heston, and D. L. Tidwell (Cedar Falls, IA: University of Northern Iowa), 14–17. //www.gov.za/sites/default/ﬁles/gcis_document/201903/national-action-plan.pdf (accessed January, 2023). //www.gov.za/sites/default/ﬁles/gcis_document/201903/national-action-plan.pdf (accessed January, 2023). Durkheim, E. (1893). The Division of Labor in Society. New York, NY: The Free Press. Araujo, M. C., Carneiro, P., Cruz-Aguayo, Y., and Schady, N. (2016). Teacher quality and learning outcomes in kindergarten. Q. J. Econ. 131, 1415–1453. Durkheim, E. (1964). The Division of Labor in Society. New York, NY: The Free Press. Future Africa Forum (2023). Low Youth Participation: A Ticking Time Bomb For Zimbabwe’s Future. Nairobi: Future Africa Forum Barolsky, V. (2016). 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Written informed consent was obtained from the individual(s) for the publication of any potentially identiﬁable images or data included in this article. Frontiers in Education 13 frontiersin.org Singh and Mukeredzi Singh and Mukeredzi Singh and Mukeredzi 10.3389/feduc.2024.1326437 Author contributions MS: Conceptualization, Data curation, Formal Analysis, Writing−original draft, Writing−review and editing. TM: Formal Analysis, Methodology, Writing−original draft. 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. Funding 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. 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https://openalex.org/W4381734950	https://www.frontiersin.org/articles/10.3389/fepid.2023.1190407/pdf	English	null	Structural racism, air pollution and the association with adverse birth outcomes in the United States: the value of examining intergenerational associations	Frontiers in epidemiology	2,023	cc-by	8,481	EDITED BY Jaime Madrigano, Johns Hopkins University, United States REVIEWED BY Jane Clougherty, Drexel University, United States Alison Teyhan, University of Bristol, United Kingdom CORRESPONDENCE Marie S. O’Neill marieo@umich.edu RECEIVED 20 March 2023 ACCEPTED 26 May 2023 PUBLISHED 22 June 2023 CITATION Buxton MA, Fleischer NL, Ro A and O’Neill MS (2023) Structural racism, air pollution and the association with adverse birth outcomes in the United States: the value of examining intergenerational associations. Front. Epidemiol. 3:1190407. doi: 10.3389/fepid.2023.1190407 EDITED BY Jaime Madrigano, Johns Hopkins University, United States REVIEWED BY Jane Clougherty, Drexel University, United States Alison Teyhan, University of Bristol, United Kingdom CORRESPONDENCE Marie S. O’Neill marieo@umich.edu RECEIVED 20 March 2023 ACCEPTED 26 May 2023 PUBLISHED 22 June 2023 CITATION Buxton MA, Fleischer NL, Ro A and O’Neill MS (2023) Structural racism, air pollution and the association with adverse birth outcomes in the United States: the value of examining intergenerational associations. Front. Epidemiol. 3:1190407. doi: 10.3389/fepid.2023.1190407 EDITED BY Jaime Madrigano, Johns Hopkins University, United States REVIEWED BY Jane Clougherty, Drexel University, United States Alison Teyhan, University of Bristol, United Kingdom CORRESPONDENCE Marie S. O’Neill marieo@umich.edu RECEIVED 20 March 2023 ACCEPTED 26 May 2023 PUBLISHED 22 June 2023 CITATION Buxton MA, Fleischer NL, Ro A and O’Neill MS (2023) Structural racism, air pollution and the association with adverse birth outcomes in the United States: the value of examining intergenerational associations. Front. Epidemiol. 3:1190407. doi: 10.3389/fepid.2023.1190407 EDITED BY Jaime Madrigano, Johns Hopkins University, United States REVIEWED BY Jane Clougherty, Drexel University, United States Alison Teyhan, University of Bristol, United Kingdom CORRESPONDENCE Marie S. O’Neill marieo@umich.edu RECEIVED 20 March 2023 ACCEPTED 26 May 2023 PUBLISHED 22 June 2023 CITATION Buxton MA, Fleischer NL, Ro A and O’Neill MS (2023) Structural racism, air pollution and the association with adverse birth outcomes in the United States: the value of examining intergenerational associations. Front. Epidemiol. 3:1190407. doi: 10.3389/fepid.2023.1190407 EDITED BY Jaime Madrigano, Johns Hopkins University, United States REVIEWED BY Jane Clougherty, Drexel University, United States Alison Teyhan, University of Bristol, United Kingdom Miatta A. Buxton 1, Nancy L. Fleischer 1, Annie Ro 2 and Marie S. TYPE Review PUBLISHED 22 June 2023 DOI 10.3389/fepid.2023.1190407 KEYWORDS structural racism, air pollution, intergenerational associations, existing data sources, adverse birth outcomes O’Neill 1,3* 1Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI, United States, 2Department of Health, Society, and Behavior, Program in Public Health, University of California, Irvine, Irvine, CA, United States, 3Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI, United States Buxton MA, Fleischer NL, Ro A and O’Neill MS (2023) Structural racism, air pollution and the association with adverse birth outcomes in the United States: the value of examining intergenerational associations. Front. Epidemiol. 3:1190407. doi: 10.3389/fepid.2023.1190407 Structurally racist policies and practices of the past are likely to be a driving factor in current day differences in exposure to air pollution and may contribute to observed racial and ethnic disparities in adverse birth outcomes in the United States (U.S.). Non-Hispanic Black women in the U.S. experience poorer health outcomes during pregnancy and throughout the life course compared to non-Hispanic White women. This disparity holds even among non-Hispanic Black women with higher socioeconomic status. Reasons for this ﬁnding remain unclear, but long-term environmental exposure, either historical exposure or both historical and ongoing exposure, may contribute. Structural racism likely contributes to differences in social and environmental exposures by race in the U.S. context, and these differences can affect health and wellbeing across multiple generations. In this paper, we brieﬂy review current knowledge and recommendations on the study of race and structural racism in environmental epidemiology, speciﬁcally focused on air pollution. We describe a conceptual framework and opportunities to use existing historical data from multiple sources to evaluate multi-generational inﬂuences of air pollution and structurally racist policies on birth and other relevant health outcomes. Increased analysis of this kind of data is critical for our understanding of structural racism’s impact on multiple factors, including environmental exposures and adverse health outcomes, and identifying how past policies can have enduring legacies in shaping health and well-being in the present day. The intended purpose of this manuscript is to provide an overview of the widespread reach of structural racism, its potential association with health disparities and a comprehensive approach in environmental health research that may be required to study and address these problems in the U.S. The collaborative and methodological approaches we highlight have the potential to identify modiﬁable factors that can lead to effective interventions for health equity. Introduction Additionally, environmental justice scholars and activists in the United States have documented that exposure to air pollution and other adverse environmental conditions are often disproportionately higher among racially minoritized groups (13), despite lower consumption of goods and services associated with the production of air pollution (14). In addition, associations have been examined between redlining and current day air pollution levels in cities across the U.S (2). Although air pollution concentrations have declined over time, disparities have persisted (4). Exposure remains a problem from an environmental justice COPYRIGHT © 2023 Buxton, Fleischer, Ro and O'Neill. 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. 01 Frontiers in Epidemiology frontiersin.org 10.3389/fepid.2023.1190407 Buxton et al. point of view because even levels below health-based regulatory standards are associated with adverse health outcomes (15, 16). Introduction A comprehensive approach to understanding factors that may contribute to disparities in adverse birth outcomes will be crucial for the development of effective intervention strategies in the U.S. Race, or the division of the population by racially deﬁned subgroups, has been described as a socially constructed undertaking that does not have a biological basis (5, 6) and may not be causally associated with disparities observed in key health outcomes such as adverse birth outcomes. Instead, policies and practices such as redlining, a government-backed practice in the 1930s that led to the classiﬁcation of neighborhoods with a high percentage of Black individuals and immigrants as “hazardous” and consequently, considered a low investment priority by lenders (2), may be a key component driving observed differences in birth outcomes. Maps created by the Home Owners’ Loan Corporation (HOLC), a government-sponsored entity intended to assist homeowners at risk of foreclosure, informed this practice. The HOLC utilized the following system to classify neighborhoods: Green = “Best”; Blue = “Still Desirable”; Yellow = “Declining”; Red = “Hazardous” (7). This practice led to disinvestment in neighborhoods that were redlined or classiﬁed as “red”, which subsequently led to major declines in neighborhood quality and lost opportunities for home ownership and wealth accumulation (8). With neighborhood decline, land value may have been affected and this may have created conditions that were favorable to industries that produce pollution (9). In addition, surrounding areas near redlined neighborhoods were zoned as “industrial”, thereby allowing for the siting of major pollution producing sources (10). Despite ofﬁcially ending in 1968 (11), redlining’s impact has persisted and links exist between redlined neighborhoods and the siting of present-day pollution sources such as roadways, industrial facilities and power plants (2, 12). Additionally, environmental justice scholars and activists in the United States have documented that exposure to air pollution and other adverse environmental conditions are often disproportionately higher among racially minoritized groups (13), despite lower consumption of goods and services associated with the Structural racism may play a role in observed disparities in adverse birth outcomes by different racial groups in the United States (U.S.). Introduction Structural racism, described as “the larger system of policies, practices, ideologies, and institutions that foster racial inequality by creating differential access to resources and opportunities” (1), is reported to be a major driver of the uneven distribution of air pollution across different racial and ethnic minoritized groups in the U.S in both the short and long term (2–4). The impact of these disparities may be even greater when consideration is given to the historical context of structural racism and corresponding data are incorporated in study designs. A comprehensive approach to understanding factors that may contribute to disparities in adverse birth outcomes will be crucial for the development of effective intervention strategies in the U.S. for the development of effective intervention strategies in the U.S. Race, or the division of the population by racially deﬁned subgroups, has been described as a socially constructed undertaking that does not have a biological basis (5, 6) and may not be causally associated with disparities observed in key health outcomes such as adverse birth outcomes. Instead, policies and practices such as redlining, a government-backed practice in the 1930s that led to the classiﬁcation of neighborhoods with a high percentage of Black individuals and immigrants as “hazardous” and consequently, considered a low investment priority by lenders (2), may be a key component driving observed differences in birth outcomes. Maps created by the Home Owners’ Loan Corporation (HOLC), a government-sponsored entity intended to assist homeowners at risk of foreclosure, informed this practice. The HOLC utilized the following system to classify neighborhoods: Green = “Best”; Blue = “Still Desirable”; Yellow = “Declining”; Red = “Hazardous” (7). This practice led to disinvestment in neighborhoods that were redlined or classiﬁed as “red”, which subsequently led to major declines in neighborhood quality and lost opportunities for home ownership and wealth accumulation (8). With neighborhood decline, land value may have been affected and this may have created conditions that were favorable to industries that produce pollution (9). In addition, surrounding areas near redlined neighborhoods were zoned as “industrial”, thereby allowing for the siting of major pollution producing sources (10). Despite ofﬁcially ending in 1968 (11), redlining’s impact has persisted and links exist between redlined neighborhoods and the siting of present-day pollution sources such as roadways, industrial facilities and power plants (2, 12). Introduction Despite increasing evidence that redlining and other important historical policies are associated with disparities in health, educational and economic outcomes, particularly given the pervasive and persistent nature of structural racism, very few studies focus on intergenerational associations (17). In environmental epidemiology, the lack of widespread use of methodological approaches that incorporate intergenerational exposures and measures of structural racism may partly be due to logistical complexity in ﬁnding and linking datasets across generations. The ﬁrst goal of this article is to provide context about studying racial disparities, structural racism and environmental epidemiology. Then, we review evidence on air pollution associations with adverse birth outcomes. In addition, we discuss a conceptual framework that informs the rationale behind our recommendation to evaluate associations between intergenerational exposure to air pollution and adverse birth outcomes. We also identify examples of existing historical data from multiple sources to evaluate these intergenerational associations and to identify factors, including structurally racist policies that differ across communities, which may provide opportunities for intervention. In this manuscript, we focus on non-Hispanic Black and non-Hispanic White persons because the largest disparities in adverse birth outcomes such as preterm birth, low birth weight and infant mortality are found among non-Hispanic Black individuals compared to non-Hispanic White individuals. However, the methods here would be applicable and equally important in studying health disparities affecting other racially minoritized groups. Ultimately, the intended purpose of this manuscript is to provide an overview of the current state of structural racism in environmental epidemiology, introduce a conceptual framework of intergenerational exposures, and offer data considerations when taking an intergenerational approach in environmental epidemiological research. Structural racism may play a role in observed disparities in adverse birth outcomes by different racial groups in the United States (U.S.). Structural racism, described as “the larger system of policies, practices, ideologies, and institutions that foster racial inequality by creating differential access to resources and opportunities” (1), is reported to be a major driver of the uneven distribution of air pollution across different racial and ethnic minoritized groups in the U.S in both the short and long term (2–4). The impact of these disparities may be even greater when consideration is given to the historical context of structural racism and corresponding data are incorporated in study designs. Frontiers in Epidemiology Studying race and structural racism and current practice in environmental epidemiology as a way of illustrating current practice and areas for improvement on the conceptualization of race in the environmental health literature (22). They found a pattern in which the reviewed studies provided little discussion conceptualizing and justifying the use of race in the study, and few discussions of reasons behind any observed effect modiﬁcation by race. Indeed, the authors suggest that frameworks that include social meaning of race and positioning of race within structural and cultural systems are lacking in the air pollution and mortality literature, and call for greater “rigor and intention” in the way race is considered in such studies. One example they provide is looking at the role of “place within race categories” as a way of identifying features that differ by place and their role in exposure/outcome differences. They also call for framing of research questions in a way that is grounded in the humanities literature and has clear policy and intervention implications. Similar calls were given in a recent editorial emphasizing interest in publishing research on environmental racism in environmental health sciences research and introducing new guidelines to enhance the rigor of such research (23). Potential links between air pollution exposure and adverse birth outcomes may lead to the perpetuation of increased risks among subsequent generations and contribute to sustained disparities across generations. For example, low birth weight and preterm birth rates are higher among individuals who had lower than normal weight at birth or who were born prior to 37 weeks of gestation (36), and intergenerational associations have been reported (37, 38). Numerous studies conducted to date have evaluated long-term or pregnancy-wide exposure to air pollution and birth outcomes within a single generation (39). Others have evaluated pre- and postnatal exposures on child health outcomes (40). However, exposure to air pollution may have health impacts across generations that may not be fully accounted for using only prenatal exposures or exposures within a given generation. Examining intergenerational air pollution exposure may be helpful for understanding racial and ethnic health disparities in subsequent generations and help account for yet unexplained racial and ethnic differences in adverse birth outcomes. Associations may operate via multiple pathways that include increased potential for morbidity during the life course, and reduced socioeconomic advancement opportunities among families. Studying race and structural racism and current practice in environmental epidemiology Many studies evaluating differences in exposure to environmental pollution or adverse birth outcomes compare a racially minoritized group or groups to non-Hispanic White persons as the reference group (18). Although useful for highlighting differences by racially minoritized groups with non- minoritized groups, continued comparisons of health outcomes by race without a discussion of the myriad of factors that race represents may lead to incorrect conclusions or perpetuation of the idea that observed disparities may be due to biological differences and thus limit potential opportunities for intervention. Instead, health disparities by racially classiﬁed categories are likely in many cases to result from longstanding exclusionary and racist systems and practices (19). Therefore, 02 Frontiers in Epidemiology frontiersin.org 10.3389/fepid.2023.1190407 10.3389/fepid.2023.1190407 Buxton et al. and ongoing stressors may operate via the “weathering hypothesis”, which posits that combined and repeated exposure to social and environmental stressors over the life course may lead to accelerated declines in health (33) and may help explain the mechanisms involved in the cumulative potential of harmful exposures. Although weathering can affect anyone who is continually exposed to stressors, including non-Hispanic White individuals, the effects are more pronounced among non- Hispanic Black individuals (34, 35). An important consideration for understanding the broader systems and processes that may contribute to persistent disparities in adverse health outcomes between non-Hispanic White and non-Hispanic Black individuals is that weathering does not account for environmental exposures or other stressors across generations, which may be important in understanding cumulative risks. Extending the weathering hypothesis by looking beyond an individual’s life course to include intergenerational exposures may be important in understanding risks in subsequent generations among non-Hispanic Black families. using rigorous study designs that illuminate the impact of these systems will ultimately inform policies needed to change these systems and will likely advance the ﬁeld in meaningful ways (20). The use of multilevel and multidimensional models (e.g., use of factor analysis to combine multiple dimensions of structural racism) (21); mixed data (e.g., individual level biological data and administrative data, or mixing of quantitative and qualitative data, interactions among structural and personal levels of racism) (17); and life course and longitudinal analyses, have been recommended as a way to move health disparities research forward (19). Speciﬁc to environmental epidemiology, Hicken and colleagues recently reviewed literature on race variables as an effect modiﬁer of the air pollution and mortality association in the U.S. Studying race and structural racism and current practice in environmental epidemiology For example, Sharkey found that individuals whose childhood residence was in the lowest quartile of income, which are disproportionately Black individuals, tended to live in similar neighborhoods as adults, suggesting that neighborhood disadvantage can be “inherited” across generations (41). Neighborhood income and racial composition, as well, are associated with higher levels of air pollution as well as potentially toxic components of pollution (42), and these disparities can persist over time and contribute to illness. Morbidity during the life course may subsequently contribute to lower socio-economic status (SES) of the mother and other caregivers at the birth of the next generation, and increased risks of adverse birth and child health outcomes in subsequent generations (Figure 1). In addition, multiple pathways may involve direct and indirect effects, including epigenetic changes in germline cells that have been reported for air pollution and other environmental Frontiers in Epidemiology frontiersin.org Air pollution and adverse birth outcomes—Conceptual framework for intergenerational studies Several individual epidemiologic studies and meta-analyses support associations between higher levels of air pollution and adverse birth outcomes (24–26). Associations are hypothesized to occur via multiple mechanisms, including oxidative stress, inﬂammation, and endothelial dysfunction (27). Air pollution is a risk factor for adverse birth outcomes, such as low birth weight (26) and preterm birth (25, 28, 29). Such birth outcomes occur at higher rates among non-Hispanic Black compared to non- Hispanic White women (30), and persist even after accounting for important factors such as education and socioeconomic status (31, 32). Reasons for this ﬁnding remain unclear, but long-term air pollution exposure, either historical or both historical and ongoing exposure to air pollution, may partly contribute. It is thus crucial for the underlying factors, including systemic factors, which affect exposure to air pollution to be investigated in any serious attempts to address health disparities in the U.S. Previous 03 frontiersin.org Buxton et al. 10.3389/fepid.2023.1190407 FIGURE 1 Multi-generational exposure to air pollution and adverse birth outcomes—conceptual framework. FIGURE 1 Multi-generational exposure to air pollution and adverse birth outcomes—conceptual framework. the same family. Birth outcomes are available in this dataset as this information is routinely collected at birth and recorded on birth certiﬁcates. A description of this multigenerational dataset for 1989–2014 has been previously reported (45) and the dataset includes 60,258 linked families (family trees), with 32,046 for non-Hispanic Black infants and 28,212 for non-Hispanic White infants. exposures (43, 44), that work together to produce adverse health outcomes in subsequent generations. Germline cells lead to the development of the reproductive cells in females and males and may lead to the transfer of altered gene expression across generations. Examples of data sources in the U.S. for examining intergenerational associations In addition to the South Carolina birth outcome data and multigenerational dataset, other states have produced similar multigenerational datasets. Two of those intergenerational datasets cover periods in the early 1980s for which census tract level air pollution estimates are available for at least a part of the older generation life span. First, using data from the State of Washington, a database was created of vital statistics data that were subsequently linked to birth certiﬁcate data of mothers born in Washington State (46). The resulting dataset included 46,000 births that occurred between 1987 and 1995 to mothers who were born between 1949 and 79 and who were of the following racial and ethnic groups: African American, Hispanic, Native American and White individuals. Second, an intergenerational dataset was generated by linking the State of Virginia live birth data from 2005 to 2009 to mothers born during 1960–1997. This overall dataset included 170,624 records, but a subset that was restricted to non-Hispanic Black and non-Hispanic White ﬁrst- born singleton infants included 69,702 records (47). Frontiers in Epidemiology frontiersin.org Health outcomes Researchers interested in conducting studies to examine intergenerational associations can beneﬁt from using existing data resources, such as linked birth registries data or existing cohorts. In other instances, opportunities may exist to work with state level or federal agencies to obtain restricted identiﬁable data. To date, a number of studies have reported analyses using intergenerational datasets; these datasets may provide opportunities to investigate the role of structural racism on environmental exposures and adverse health outcomes. One example of such a dataset that provides potential opportunities to investigate intergenerational associations is the South Carolina birth outcome data and multigenerational dataset. This is an integrative dataset that includes data obtained from singleton birth certiﬁcates for the state of South Carolina from 1989 to 2014 (45). Birth data are geocoded at the census tract level and linked along the maternal line and span three generations. In the U.S., census tracts are statistical subdivisions of varying geographical area that include an average population size of 4,000 inhabitants, range 1,200–8,000 inhabitants (U.S Census Bureau). The resulting structure of the data includes grandmothers (G1), mothers (G2), and G2’s children (G3) within In addition, a number of multigenerational studies (48, 49), have been established and may serve as either potential data sources or methodological inspiration for future studies. Furthermore, future studies exploring the logistical complexity of assembling a cohort of three generations could incorporate the design of the Nurses’ Health Study II (50). In the Nurses’ Health Study II, both historical and current data were collected, thereby enabling the utilization of data on three generations. Speciﬁcally, enrolled participants represent the second generation or G2 and 04 frontiersin.org Buxton et al. 10.3389/fepid.2023.1190407 10.3389/fepid.2023.1190407 information was gathered on G1 and G3. These datasets point to existing opportunities and highlight methodology that may be applied to different states with other characteristics of interest. The CACES website has air pollution data that can be easily requested in a few steps, the process does not require a lengthy processing time, and the data timeframe supports examination of intergenerational exposures. Data availability from CACES varies across pollutants, but the earliest year is 1979, which would make this data source compatible with studies interested in at least two generations. This data source is particularly useful for studies interested in average exposure as the data are available as annual estimates. CACES focuses on key themes about air pollution, including regional differences of air pollution. Health outcomes Additionally, CACES incorporates measurement and modeling approaches that are helpful for investigating both spatial and temporal differences in exposure and associated outcomes. Information on CACES and their air pollution data may be accessed at the following website (www.caces.us/data). Data available at CACES can be used to estimate annual air pollution exposure for individual participants at the census tract level. CACES produces air pollution estimates for criteria air pollutants, the air pollutants for which the U.S. EPA has established standards to protect human health. Criteria pollutants include carbon monoxide (CO), ozone (O3), particulate matter ≤10, ≤2.5 μm in aerodynamic diameter (PM10, PM2.5), nitrogen dioxide (NO2), and sulfur dioxide (SO2). According to CACES, average annual estimates, except for ozone, were derived using models that incorporate data from U.S. EPA regulatory monitors, satellite-derived air pollution estimates of air pollution to account for locations with missing data, and information on land use. For ozone, estimates are based on the daily maximum 8-hour moving average concentrations for the period May through September (54), which corresponds to the warmer months of the year (55). Figure 2 shows a sample of air pollution data obtained from CACES using PM10 from 1989 to 2015 for two census tracts in South Carolina. The census tracts shown are in Allendale [Federal Information Processing Standards (FIPS) code: 45005970200] and Oconee (FIPS code: 45073030602). These census tracts were selected to compare a census tract with a high percent of non-Hispanic White population to a high percent non-Hispanic Black population. According to 2010 census data available on Social Explorer (https://www.socialexplorer.com/ explore-tables), which provides access to current and historical U.S census data, the population for the census tract in Allendale was 78.4% Black and the population for the census tract in Oconee was 88.4% White. These data can be generated for any census tract in the United States and annual concentrations of air pollutants can be compared over time. Data are available for O3, SO2, NO2, for 1979–2017, PM10 for 1988–2017, CO 1990– 2017 and PM2.5 from 1999 to 2017. Another source of historical air pollution data is from the The intergenerational data format described here offers an advantage over single point-in-time measures of structural racism or environmental exposures and may elucidate pathways leading to adverse health outcomes. Health outcomes Using intergenerational information may inform whether maternal exposure to structural racism impacts health outcomes through shared exposure (i.e., inherited disadvantage) or through biological pathways (i.e., a mother’s birthweight is a determinant of her offspring’s birthweight). An important consideration is the statistical approach that would be appropriate for intergenerational studies and effectively account for both historical and ongoing exposures. Mediation analyses have been previously used to understand direct and indirect effects in studies examining smoking transmission across three generations (51, 52), and such an approach would be useful for studies that might be conducted with the data described here. For example, in one study that examined smoking transmission across three generations over a 45-year period, the study team utilized a multi- step approach (52). First, logistic regression models were utilized to evaluate direct relationships for each of the following pairs: associations between the ﬁrst generation (G1) and second generation (G2) smoking, G2 and the third generation (G3) smoking, and G1 and G3 smoking. Second, the investigators examined whether transmission of smoking between the grandparents (G1) and grandchildren (G3) was mediated by G2 smoking using a Sobel test that incorporated standardized coefﬁcients resulting from the logistic regression models. Another approach could be the use of multilevel logistic regression to account for clustering resulting from the use of census tract air pollution data. Pollutant may be represented by typologies that could be used to create a composite measure of air pollution for G1 and G2. Based on the distribution of the data, a cutoff value could be used to create high and low categories for G1 and G2, separately. Combining the resulting data would form the following categories G1 high/G2 high, G1 high/G2 low, G1 low/G2 high, and G1 low/G2 low (45). In addition, the large dataset afforded by using available birth data over a long period would allow for examination of the joint effects of G1 and G2 prenatal exposure to air pollution on adverse birth outcomes among G3. Frontiers in Epidemiology frontiersin.org Air pollution The United States Environmental Protection Agency (U.S EPA) routinely collects data on air pollution. The U.S EPA compiles data from states reporting hourly air pollution concentrations at approximately 5,000 monitors across the country (53) and these historical data (currently available from 1980 to 2022) are publicly available to be linked to existing or even newly created intergenerational datasets. Additionally, other groups have compiled EPA and other data using modeling. For example, the Center for Air, Climate, and Energy Solutions (CACES) is a research center staffed by researchers from multiple disciplines and institutions. Another source of historical air pollution data is from the Atmospheric Composition Analysis Group at Washington University in St. Louis. This source has monthly and annual measures for ﬁne particulate matter which are available beginning from 1981 (56). This data source is particularly useful for studies examining time varying exposures and outcomes that might be impacted by windows of susceptibility that vary over time because it includes data at a ﬁner temporal scale (monthly estimates) compared to the CACES data (annual estimates). The Frontiers in Epidemiology 05 frontiersin.org Buxton et al. 10.3389/fepid.2023.1190407 FIGURE 2 Annual average concentrations of PM10(μg/m3) 1988–2015. FIGURE 2 Annual average concentrations of PM10(μg/m3) 1988–2015. spatial resolution is 1 km by 1 km grid squares. Other sources of historical air pollution data that allow for evaluating time varying exposures and windows of susceptibility over the gestational period are also publicly available. For example, using data from the U.S EPA Air Quality System (AQS) and other publicly available data sources including meteorological and land use data, daily air pollution data for different periods of gestation can be estimated at the census tract level based on methods used in long term studies of air pollution (57). Data from the EPA AQS can be used to estimate daily prenatal exposure (versus the average annual concentrations in the CACES data). This provides a distinct advantage in that it accounts for variations in pollutant levels at different points in the year as well as potential windows of susceptibility during pregnancy, such as trimester speciﬁc exposure, or exposures from shorter times frames, such as weeks or days before delivery (58). Methods to assign exposure estimates of census tract-level concentrations are described in Di et al. (57). Air pollution In addition to recommendations to use multilevel data and incorporate the life course and time, and, as we have discussed here, across generations, a major advantage of using such datasets, especially birth registry data, is that they are sufﬁciently large and adequately powered to allow for the evaluation of effect modiﬁcation and subgroup analyses. individuals who live miles away from a monitor if using census tract level estimates. Modelling, which takes into account other information such as land use and satellite data (54), is used in some cases and may improve estimates. However, historical data do not take into account daily activity patterns (i.e., time spent outside vs. inside) as has been done in prospective studies (59) or adjust for time that individuals may be at locations other than at home (60). In other cases, kriging, an interpolation method that takes into account available data, including address information and distance to available monitors, to improve air pollution estimates, may be used (61). Third, these limitations may inﬂuence study results particularly if the misclassiﬁcation varies by groups and caution should be taken when interpreting and discussing results. Nevertheless, utilizing the above data sources will provide unique opportunities to examine factors that may contribute to health disparities. Equally important, studies using data sources listed above may be important in identifying gaps for future prospective studies where air pollution exposure assessments may be improved. Frontiers in Epidemiology Summary Combining long-term air pollution and other environmental data and linked multi-generational health outcome data is essential for environmental epidemiologists interested in estimating associations between air pollution and intergenerational social, health and economic disparities. Further, evaluating pathways by which environmental and structural racism could play a role in these associations, and incorporating available metrics that reﬂect the historical underpinnings of the systems in our society that result in unfair exposures and health burdens, has been called for on multiple fronts. Insights obtained from this kind of study may be useful for understanding Although the historical air pollution data sources listed above are, in some cases, readily available, they are not without limitation. First, attempting to document air pollution exposure over long periods of time is a challenge. Challenges include availability of data to cover the entire timeframe of interest or if using data from multiple sources, potential differences in measurement methodology might be an issue. Second, an important limitation is that air pollution estimates using only data from the U.S EPA may be based on monitors that are placed in a central location and the same estimates may be assigned to individuals who live near a sited monitor and to 06 frontiersin.org frontiersin.org Buxton et al. 10.3389/fepid.2023.1190407 10.3389/fepid.2023.1190407 for providing a comprehensive understanding of the cumulative nature of environmental exposures and provide actionable information on how public health policy and action can be tailored. persistently observed disparities in adverse birth outcomes, and intervening to eliminate them. for providing a comprehensive understanding of the cumulative nature of environmental exposures and provide actionable information on how public health policy and action can be tailored. Conﬂict of interest 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. 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. Author contributions A fundamental component to advancing health equity is a need to design rigorous research studies that quantify structural racism such that it accurately captures the multi-dimensional way it affects the lives of minoritized individuals. The data sources presented here provide opportunities to examine associations between exposure to air pollution, which may be driven by structurally racist policies and practices, and adverse birth outcomes. These opportunities can be expanded to include maternal outcomes as well as other outcomes that have a persistent disparity between minoritized and non-minoritized groups. In addition, these steps highlight a need to focus less on individual-level factors associated with adverse outcomes and more on systematic attempts at correcting the systems that enable structurally racist practices to ﬂourish. MB, MO, NF, and AR: contributed to the conceptualization, writing-original draft, editing, funding acquisition, and methodology of this article. All authors contributed to the article and approved the submitted version. Funding This project was supported by the following sources: National Institute on Minority Health and Health Disparities grant R01 MD016046, National Institute of Environmental Health Sciences grants R01 ES016932, R01 ES017022, T32 ES007062, and P30 ES017885; National Institute for Occupational Safety and Health grant T42 OH008455-09. Consistent with recent calls to enhance the rigor and care with which hypotheses around race as an effect modiﬁer in environmental epidemiology are developed and ﬁndings explained (22, 23), we suggest that researchers can tap into existing data and frame their research in a way that points toward policy and interventions to reduce health disparities. This suggestion is consistent with a recent exhortation to move beyond characterizing race/ethnic disparities in air pollution to addressing them (62). Others have called for combining antiracist principles with those of community-based participatory research, an approach that emphasizes community knowledge and equal partnerships with academics (63). Collaborating in research with affected communities and co-analysis of collected data will lead to enhanced insights into causal mechanisms being explored (64). Thus, in addition to tapping into existing data sources and applying methods such as those we illustrate here, we advocate for engaging community members and practitioners in evaluating and framing research questions. Such approaches may be useful 1. Williams DR, Lawrence JA, Davis BA. Racism and health: evidence and needed research. Annu Rev Public Health. (2019) 40:105–25. doi: 10.1146/annurev- publhealth-040218-043750 7. Hillier AE. Who received loans? Home Owners’ loan corporation lending and discrimination in Philadelphia in the 1930s. J Plan Hist. (2003) 2(1):3–24. doi: 10. 1177/1538513202239694 Frontiers in Epidemiology 2. 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https://openalex.org/W3131125107	https://link.springer.com/content/pdf/10.1007/s10853-021-05827-z.pdf	English	null	Determining elastic anisotropy of textured polycrystals using resonant ultrasound spectroscopy	Journal of materials science	2,021	cc-by	13,335	Determining elastic anisotropy of textured polycrystals using resonant ultrasound spectroscopy Determining elastic anisotropy of textured polycrystal using resonant ultrasound spectroscopy Jordan A. Evans1, Blake T. Sturtevant1 , Bjørn Clausen1 , Sven C. Vogel1 , Fedor F. Balakirev1 , Jonathan B. Betts1, Laurent Capolungo1 , Ricardo A. Lebensohn1 , and Boris Maiorov1,* Jordan A. Evans1, Blake T. Sturtevant1 , Bjørn Clausen1 , Sven C. Vogel1 , Fedor F. Balakirev1 , Jonathan B. Betts1, Laurent Capolungo1 , Ricardo A. Lebensohn1 , and Boris Maiorov1,* Jordan A. Evans1, Blake T. Sturtevant1 , Bjørn Clausen1 , Sven C. Vogel1 , Fedor F. Balakirev1 , Jonathan B. Betts1, Laurent Capolungo1 , Ricardo A. Lebensohn1 , and Boris Maiorov1,* 1Los Alamos National Laboratory, Los Alamos, NM 87545, USA 1Los Alamos National Laboratory, Los Alamos, NM 87545, USA Received: 27 August 2020 Accepted: 20 January 2021 Published online: 24 February 2021 This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2021 METALS & CORROSION J Mater Sci (2021) 56:10053–10073 Metals & corrosion METALS & CORROSION J Mater Sci (2021) 56:10053–10073 Metals & corrosion J Mater Sci (2021) 56:10053–10073 Handling Editor: P. Nash. Address correspondence to E-mail: maiorov@lanl.gov Received: 27 August 2020 Accepted: 20 January 2021 Published online: 24 February 2021 This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2021 METALS & CORROSION Metals & corrosion Determining elastic anisotropy of textured polycrystals using resonant ultrasound spectroscopy Jordan A. Evans1, Blake T. Sturtevant1 , Bjørn Clausen1 , Sven C. Vogel1 , Fedor F. Balakirev1 , Jonathan B. Betts1, Laurent Capolungo1 , Ricardo A. Lebensohn1 , and Boris Maiorov1,* Introduction Most polycrystalline materials show some degree of anisotropy. In general, anisotropy may arise in these materials from the crystal symmetry of the material compounded with a distribution of crystallographic orientations of the single-crystal grains in the aggre- gate (i.e., texture), and/or by an inhomogeneous distribution of secondary phases [1]. Defects gener- ated by thermomechanical processes can also pro- duce elastic anisotropy if they exhibit a preferential alignment or orientation [2]. Anisotropic properties have profound inﬂuence on many properties, such as a material’s response to mechanical or magnetic stimuli, and can be exploited to design materials with directionally preferential properties [3–5]. Crystallographic texture can result from a variety of common manufacturing and forming processes, such as extruding, swaging, rolling and cold working [6–12]. Texture can be characterized by several dis- tinct methods, including X-ray diffraction (XRD), electron backscatter diffraction (EBSD) and neutron scattering [4, 13–18]. However, these methods are either limited to characterizing texture on the surface of the material, or require large user facilities with competitive access. The objective of the present work is to assess the potential for RUS to identify the symmetry and ori- entation of the anisotropy of a polycrystalline alu- minum sample, as well as to quantify the degree of anisotropy. The focus of the present work is placed on indirectly quantifying the symmetry of the crystal orientation distribution rather than providing a full texture representation. Aluminum is an inherently low-anisotropy material and was selected with the intention of testing the lower limits of detection possible using RUS. Application of the RUS method on extruded aluminum samples clearly suggests that the material texture is transversely isotropic. p Alternatively, acoustic characterization techniques offer the possibility of probing the bulk of a material as mechanical waves travel through it, as is the case for the pulse-echo ultrasonic technique [19]. In par- ticular, resonant ultrasound spectroscopy (RUS) is a fast, high-resolution, nondestructive characterization technique whereby the mechanical resonance fre- quencies of a specimen can be measured and used to determine the elastic properties of a material. RUS, nonlinear RUS and pulse-echo characterization con- stitute a powerful arsenal of nondestructive charac- terization techniques [20, 21]. The mechanical resonance frequencies of a material are set by the sample’s geometry, mass, internal symmetry, and elastic constants. Using these parameters, one can obtain the complete elastic tensor by solving the inverse problem [22]. ABSTRACT Polycrystalline materials can have complex anisotropic properties depending on their crystallographic texture and crystal structure. In this study, we use reso- nant ultrasound spectroscopy (RUS) to nondestructively quantify the elastic anisotropy in extruded aluminum alloy 1100-O, an inherently low-anisotropy material. Further, we show that RUS can be used to indirectly provide a description of the material’s texture, which in the present case is found to be transversely isotropic. By determining the entire elastic tensor, we can identify the level and orientation of the anisotropy originated during extrusion. The relative anisotropy of the compressive (c11/c33) and shear (c44/c66) elastic con- stants is 1.5% ± 0.5% and 5.7% ± 0.5%, respectively, where the elastic constants (ﬁve independent elastic constants for transversely isotropic) are those associ- ated with the extrusion axis that deﬁnes the symmetry of the texture. These results indicate that the texture is expected to have transversely isotropic symmetry. This ﬁnding is conﬁrmed by two additional approaches. First, we conﬁrm elastic constants and the degree of elastic anisotropy by direct sound velocity measurements using ultrasonic pulse echo. Second, neutron diffraction (ND) data conﬁrm the symmetry of the bulk texture consistent with extrusion- induced anisotropy, and polycrystal elasticity simulations using the elastic self- consistent model with input from ND textures and aluminum single-crystal elastic constants render similar levels of polycrystal elastic anisotropy to those measured by RUS. We demonstrate the ability of RUS to detect texture-induced anisotropy in inherently low-anisotropy materials. Therefore, as many other common materials have intrinsically higher elastic anisotropy, this technique should be applicable for similar levels of texture, providing an efﬁcient general diagnostic and characterization tool. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2021 10054 J Mater Sci (2021) 56:10053–10073 texture. However, the symmetry selection process and the results obtained were not fully validated against texture measurements from other methods [28, 29]. Foster et al. [30] used RUS to determine the elastic anisotropy of rolled Cu–Zn (brass) alloys in combination with analytical expressions derived from polycrystal models based on simple averaging procedures to obtain a relatively compact represen- tation of the sample’s texture in terms of principal orientation distribution coefﬁcients for comparison with texture measurements by neutron diffraction (ND). A related approach, but using new RUS and ND measurements on Al polycrystalline samples and more sophisticated modeling tools, is used in this work. ABSTRACT More recently, Lan et al. [28, 29] proposed a new technique to directly measure the texture of a material using acoustic waves and contrasted this technique against resonant ultrasound spectroscopy (RUS) and neutron diffraction. Similarly, the effect of density of defects has been studied by multiple ultrasonic and microstructural characterization methods in aluminum and other material [20, 21]. Introduction This can be accomplished by using a nonlinear iterative Levenberg–Marquardt algorithm, which minimizes the deviation between the measured and predicted values of resonance frequencies based on the material’s elastic constants [23–27]. Historically, there have been attempts to use RUS to quantify the elastic anisotropy induced by Further, we validate RUS results with direct mea- surements using ultrasonic pulse-echo technique, as well as crystallographic texture measurements by neutron diffraction combined with elastic self-con- sistent (ELSC) micromechanical simulations that give the polycrystal elastic constants as a function of the known single-crystal elastic constants and the texture of the sample. The ultrasonic pulse-echo technique (PE) is another method that can be used to determine the elastic properties of a material. In PE, the sound velocity of the material is measured along different orientations [31–33]. Because the sound velocity is extracted directly from the time-of-ﬂight, PE results are not subject to ﬁtting or convergence. For lower- symmetry materials, however, PE cannot determine 10055 J Mater Sci (2021) 56:10053–10073 all of the elastic constants in a single measurement. Independent measurements in different orientations with respect to the anisotropy of the sample are required [34, 35]. The consequent sample manipula- tion may introduce variations in delicate samples, or result in complications when characterizing haz- ardous samples. These ultrasonic experiments are performed in the anelastic limit, i.e., the response is elastic (linear) and non-hysteretic. The level of losses is minimal but measurable, allowing RUS and PE the capability to extract changes in sound attenuation (internal friction). As a result, the measurements are nondestructive and samples are unchanged. PE measurements are found to be in very good agree- ment with RUS characterization. Finally, the ELSC predictions of the material’s effective stiffness tensor, which utilizes the materials’ bulk texture as mea- sured by neutron diffraction, shows very good, but not perfect, agreement with those measured by RUS, thereby demonstrating that the RUS can be used to extract key features in the elastic properties of strongly textured materials. case, the stiffness tensor only has two independent constants, i.e., two degrees of freedom (DoF), and reduces to Eq. 3 where c12 = c11–2c44. For materials with cubic symmetry, the stiffness tensor has three independent elastic constants, whereby c12 is inde- pendent of c44 and c11. Cij ¼ c11 c12 c12 0 0 0 c11 c12 0 0 0 c11 0 0 0 c44 0 0 sym c44 0 c44 2 6666664 3 7777775 ð3Þ ð3Þ The effective values of c11 and c44 for a polycrys- talline material depend on the single-crystalline counterparts. There are different approximations to calculate this relationship, the most common of which are the Voigt [36, 37] and Reuss [36, 37] aver- ages obtained by assuming homogenous strain or stress, respectively, which, respectively, provide the upper and lower bounds for the polycrystal’s elastic constants estimates. Alternatively, the Hashin and Shtrikman method which is based on a variational approach can also provide bounds [38, 39]. Finally, the elastic self-consistent (ELSC) model, originally proposed by Hershey [40] for aforementioned case of polycrystalline aggregates of randomly oriented sin- gle crystals with cubic symmetry, can provide a unique estimate of the homogenized elastic response of the medium [40]. These models can be generalized to aggregates of arbitrary single-crystal symmetry and non-uniform texture, in which case the poly- crystal elastic constants will depend of the single- crystal anisotropy and the orientation distribution of the grains. The recent advances obtained in mathe- matical methods used to extract the crystallographic texture from ultrasonic response, even for hexagonal symmetries, make determining the elastic anisotropy even more relevant [41]. Elastic response of a solid, anisotropy and relationship to wave velocities The effective elastic response of a solid, as described by Hooke’s law in Eq. 1, linearly relates the Cauchy stress, rij, to the elastic strain, ekl, by the elastic stiff- ness tensor cijkl. In the most general anisotropic case, the stiffness tensor has 21 independent constants due to the symmetries associated with stresses and strains and the uniqueness of strain potential energy. Alter- natively, Hooke’s law can be written in matrix form using Voigt notation in Eq. 2: rij ¼ cijklekl cijkl ¼ cjikl ¼ cijlk ¼ cjilk ¼ cklij ð1Þ r11 r22 r33 r23 r13 r12 2 6666664 3 7777775 ¼ c11 c12 c13 c14 c15 c16 c22 c23 c24 c25 c26 c33 c34 c35 c36 c44 c45 c46 sym c55 c56 c66 2 6666664 3 7777775 e11 e22 e33 2e23 2e13 2e12 2 6666664 3 7777775 ð2Þ ð1Þ A measure of intrinsic anisotropy in cubic materi- als is given by Zener’s elastic anisotropy factor, A, in Eq. 4 [42]. Almost all crystalline materials have A = 1; when those materials form polycrystalline aggregates, texture means that their physical prop- erties are orientation-dependent. Aluminum has a cubic crystal structure with a relatively small Zener factor of A = 1.22 [43]. Aluminum is notably less anisotropic than most other cubic metals, such as copper (A = 3.21) [44] and 304 stainless steel (SS) (A = 3.77) [45], for example. ð2Þ For polycrystalline materials with no texture, each grain is randomly oriented such that the bulk elastic properties are equal in all directions. In this isotropic 10056 J Mater Sci (2021) 56:10053–10073 A ¼ 2c44 c11 c12 ð4Þ l23 ¼ l13 ¼ c44 l12 ¼ c66 ¼ 1 2 c11 c12 ð Þ ð12Þ m? ¼ c12c33 c2 13 c11c33 c2 13 mjj? ¼ c11c13 c12c13 c2 11 c2 12 m?jj ¼ c11c13 c12c13 c11c33 c2 13 ð13Þ l23 ¼ l13 ¼ c44 l12 ¼ c66 ¼ 1 2 c11 c12 ð Þ ð12Þ m? ¼ c12c33 c2 13 c11c33 c2 13 mjj? Elastic response of a solid, anisotropy and relationship to wave velocities ¼ c11c13 c12c13 c2 11 c2 12 m?jj ¼ c11c13 c12c13 c11c33 c2 13 ð13Þ A ¼ 2c44 c11 c12 A ¼ 2c44 c11 c12 ð4Þ ð12Þ For isotropic materials (A = 1), the Young’s mod- ulus E (stiffness under uniaxial tension), bulk mod- ulus B (resistance to volumetric changes), shear modulus l (stiffness under shear deformation) and Poisson ratio m (ratio of transversal strain to axial strain), also referred to as the engineering constants, are given by the relations in Eqs. 5, 6, 7, and 8. ð13Þ E ¼ c2 11 þ c11c12 2c2 12 c11 þ c12 ð5Þ B ¼ c11 þ 2c12 3 ð6Þ l ¼ c44 ð7Þ m ¼ c12 c11 þ c12 ð8Þ E ¼ c2 11 þ c11c12 2c2 12 c11 þ c12 ð5Þ B ¼ c11 þ 2c12 3 ð6Þ l ¼ c44 ð7Þ m ¼ c12 c11 þ c12 ð8Þ E ¼ c2 11 þ c11c12 2c2 12 c11 þ c12 ð5Þ B ¼ c11 þ 2c12 3 ð6Þ l ¼ c44 ð7Þ m ¼ c12 c11 þ c12 ð8Þ The relationships between the phase velocities of ultrasonic plane waves and the elastic constants of a material can be determined as shown in detail in the Appendix. For isotropic materials (two independent elastic constants), c44 = qvT 2 and c11 = qvL 2, where q is the density, vT is the transverse wave velocity, and vL is the longitudinal wave velocity. For materials with lower symmetries, such as the transversely isotropic case (ﬁve independent elastic constants), determining the relationships between sound velocities and elastic constants is far more complicated, as shown in the Appendix. ð5Þ ð6Þ ð8Þ If a manufacturing or forming process produces texture with axial anisotropy, as is the case for extrusion, the material will have transversely iso- tropic symmetry [46–50]. In the transversely isotropic case, the stiffness tensor has ﬁve independent con- stants (i.e., the same number than in a hexagonal symmetry), as shown in Eq. 9 and the orientation has been chosen such that the ‘‘z’’ axis is parallel to the texture axis (parallel to the rod’s extrusion direction). As a result of the transversely isotropic texture, the values of E, B, l and m depend on orientation. Sample preparation As a result of the transversely isotropic texture, the values of E, B, l and m depend on orientation. The engineering constants for a transversely isotropic solid parallel (\|\|) and perpendicular (\) to the axis of symmetry are given in Eqs. 10, 11, 12 and 13 [51–53]. All of the textured samples were machined from the same extruded rod of the commercial aluminum alloy (AA) 1100. AA-1100, also known as commer- cially pure aluminum, is composed of [ 99% alu- minum with silicon and iron as its primary impurities. The AA-1100 rod was provided in the annealed condition with no cold working (type O temper) in accordance with ASTM B221-14 standards. The AA-1100-O rod is assumed to have transversely isotropic texture as a result of the extrusion process during manufacturing [6–8]. An illustration of the transversely isotropic AA-1100-O rod is shown in Fig. 1b from whence rectangular parallelepiped res- onators (RPRs) were machined with sides a, b, or c parallel to the rod axis. RPRs are advantageous for RUS experiments in comparison with other geome- tries (cylinders, cubes or spheres) because they have no degenerate resonance modes when the dimen- sions are correctly chosen [22, 27]. The ﬂat parallel faces in principal directions also make RPRs suit- able for pulse-echo experiments. Several RPRs were All of the textured samples were machined from the same extruded rod of the commercial aluminum alloy (AA) 1100. AA-1100, also known as commer- cially pure aluminum, is composed of [ 99% alu- minum with silicon and iron as its primary impurities. The AA-1100 rod was provided in the annealed condition with no cold working (type O temper) in accordance with ASTM B221-14 standards. cij ¼ c11 c12 c13 0 0 0 c11 c13 0 0 0 c33 0 0 0 c44 0 0 sym c44 0 1 2 c11 c12 ð Þ 2 66666664 3 77777775 ð9Þ E? ¼ c2 11c33 þ 2c2 13c12 2c2 13c11 c2 12c33 c11c33 c2 13 Ek ¼ c11c33 þ c12c33 2c2 13 c11 þ c12 ð10Þ B ¼ 2c11 þ c33 þ 2 2c13 þ c12 ð Þ 9 ð11Þ ð9Þ E? Elastic response of a solid, anisotropy and relationship to wave velocities The engineering constants for a transversely isotropic solid parallel (\|\|) and perpendicular (\) to the axis of symmetry are given in Eqs. 10, 11, 12 and 13 [51–53]. J Mater Sci (2021) 56:10053–10073 10057 Figure 1 a, c RUS experimental setup with sample. b Illustration of 1100 rod with bz-axis chosen parallel to the extrusion-induced axis of texture and orientations of RPR samples machined from the rod relative to the texture axis. Figure 1 a, c RUS experimental setup with sample. b Illustration of 1100 rod with bz-axis chosen parallel to the extrusion-induced axis of texture and orientations of RPR samples machined from the rod relative to the texture axis. Figure 1 a, c RUS experimental setup with sample. b Illustration of 1100 rod with bz-axis chosen parallel to the extrusion-induced axis of texture and orientations of RPR samples machined from the rod relative to the texture axis. machined from this rod with dimensions shown in Fig. 1b, where a = 8.4 mm, b = 9.2 mm and c = 13.5 mm with a precision of 0.0006 mm. To sim- plify distinguishing the texture orientation of the samples machined, we deﬁne bz as the direction par- allel to the axis of extrusion. All samples were machined by electrical discharge machining (EDM) using skim cut settings in order to minimize surface roughness and damage caused by machining [54–56]. The AA-1100-O RPRs were then heat-treated at 615 K for 1 h in accordance with ASM standards in order to anneal defects present in the sample [57]. The AA- 1100-O RPRs have a density of 2.6899 ± 0.0034 g cm-3, calculated from measuring geometrical volume and mass using a high-precision micrometer and scale. Thus, these samples are 99.26% dense, assuming a theoretical density of 2.71 g cm-3. response signal from the other transducer. The RUS software was developed at Los Alamos National Laboratory and is freely available online [58]. The driving and detecting transducers are identical and are composed of an aluminum housing ﬁlled with epoxy, a coaxial SMA connector on the one end, and a 5-mm-diameter lead zirconate titanate (PZT) piezoelectric transducer glued to the ﬂat portion of an alumina hemisphere as described elsewhere and shown in Fig. 1a, c [59–61]. The alumina hemispher- ical cap not only provides electrical insulation for the piezoelectrics when measuring metallic samples, but also protects the piezoelectrics from physical damage while simultaneously guaranteeing excellent point contact with the sample. This allows the sample to resonate freely without having to balance it precari- ously by its corners. J Mater Sci (2021) 56:10053–10073 It also allows to reproduce the position of the sample with respect to the transduc- ers. The resonance frequencies of the specimens are independent of the positioning or orientation of the sample between the driving and detecting transduc- ers; in practice, however, some resonances may have undetectably low amplitudes which depend on positioning (e.g., if the detecting transducer is in contact with the sample which coincides with the node of a particular resonance). We found that plac- ing the sample vertically between the transducers (i.e., parallel to the c dimension) allowed the Sample preparation ¼ c2 11c33 þ 2c2 13c12 2c2 13c11 c2 12c33 c11c33 c2 13 Ek ¼ c11c33 þ c12c33 2c2 13 c11 þ c12 ð1 B ¼ 2c11 þ c33 þ 2 2c13 þ c12 ð Þ 9 ð1 ð10Þ ð11Þ Figure 1 a, c RUS experimental setup with sample. b Illustration of 1100 rod with bz-axis chosen parallel to the extrusion-induced axis of texture and orientations of RPR samples machined from the rod relative to the texture axis. Ultrasonic pulse-echo The conventional pulse-echo time-of-ﬂight technique was used to determine the speed of compressive and shear ultrasonic waves along all principal axes of the samples in order to directly obtain elastic constants for validation purposes [34, 35]. The experimental setup consisted of an arbitrary function generator and digitizing oscilloscope connected to an ultrasonic transducer, fused silica buffer rod and sample as described elsewhere [62, 63]. The electrical signal applied to the transducer was a tone burst with a Tukey envelope applied to mitigate spurious high- frequency effects [62, 64]. For improved accuracy in the determined time-of-ﬂight, measurements were performed over a range of frequencies, typi- cally * 10 MHz and * 20 MHz for shear and com- pressive modes, respectively. Measurement over a range of frequencies enables a frequency-dependent correction to be applied to account for the medium used to couple the buffer rod to the sample as pre- viously described [62]. The accuracy of measured time-of-ﬂight (s) and physical dimensions (d) and thus the sound speeds (v = 2d/s) were of order 10–4– 10–3. The RUS inverse problem ﬁtting procedure was executed using 12-order polynomials for all ﬁts, although no signiﬁcant differences were obtained using 14-order polynomials (deviations smaller than 0.02%). The results obtained in the RUS ﬁts are insensitive to the initial input elastic constant values. We used more than 30 of the ﬁrst 35 resonance fre- quencies in the ﬁtting procedure for each sample [24]. The ﬁts assume a particular symmetry, set by the degrees of freedom and orientation selected in the ﬁt [22, 24]. The ability to extract the elastic constants of a polycrystalline material requires matching the sym- metry of the ﬁt with that of the sample. Extruded aluminum has transversely isotropic texture, which Figure 2 Measured RUS spectrum for an AA-1100-O RPR with a magniﬁed view of the resonance at 274.3 kHz and the Lorentzian ﬁt overlaid. Because the same samples were used for the RUS and the PE experiments, and the transducer must be adhered to a ﬂat surface in the PE experiments, PE experiments could only be performed for h = 0 and h = 90 relative to bz (see Appendix). With this ori- entation constraint and using Eq. Resonant ultrasound spectroscopy The RUS system was constructed at Los Alamos National Laboratory, the general description of which is described elsewhere [24, 58]. In Fig. 1a, c, we can observe the experimental setup used. The RUS sys- tem interfaces with the computer via a ﬁeld-pro- grammable gate array-based Red Pitaya detector system, which is programmed to generate the exci- tation signal to one transducer as well as record the 10058 J Mater Sci (2021) 56:10053–10073 results in hexagonal symmetry (in the single-crystal sense). In order to distinguish physical orientations of the sample a, b and c (as described in the previous section and illustrated in Fig. 1b with the ﬁt orienta- tions involved in the RUS algorithm, we deﬁne the orientation of a hexagonal axis of symmetry in the ﬁt to be h. The ﬁt should only be successful if its ori- entation matches the physical orientation of the sample’s anisotropy (i.e., h\|\|bz for transverse iso- tropy). Similarly, as shown later in the paper, the symmetry and orientation can be determined by solving the inverse problem with a lower symmetry. detection of at least 30 of the ﬁrst 35 resonances. This sample arrangement was used for all the samples in order to reduce the small variations associated with measuring resonances in different positions on the sample. This arrangement, combined with the opti- mized sample geometry, yields easily detectable/ distinguishable resonances with large contributions from shear, dilatational and compressive elastic constants. In turn, the extracted resonance frequen- cies result in an overdetermined problem for the extraction of all the elastic constants from the mea- sured resonance frequencies. The quality factors (Q- values, the frequency-to-bandwidth ratio, deﬁned by the resonance frequency divided by its full-width at half-maximum) of the resonances vary, but most resonances were measured with Q-values exceeding 20,000 as shown in Fig. 2. Ultrasonic pulse-echo 21, the PE experi- ments were used to determine c11, c33, c44 and c66, while c13 could not be measured since its only con- tribution to the sound velocity comes from the sin22h component of Eq. 20 (see the Appendix). In order to measure c13 in rectangular parallelepiped samples machined from this textured rod, the faces of the Figure 2 Measured RUS spectrum for an AA-1100-O RPR with a magniﬁed view of the resonance at 274.3 kHz and the Lorentzian ﬁt overlaid. Neutron diffraction Aluminum is an inherently low-anisotropy material (A = 1.22). As such, the degree of texture required (measured in multiple of random density (MRD)) in order to result in measurable changes in its elastic properties is expected to be large. Bulk extrusion- produced texture was measured using time-of-ﬂight neutron diffraction on the high-pressure preferred orientation time-of-ﬂight diffractometer (HIPPO) [65, 66] at the pulsed spallation neutron source at the Los Alamos Neutron Science Center (LANSCE) of Los Alamos National Laboratory [67]. HIPPO utilizes 1200 3He detector tubes arranged on 45 detector panels on ﬁve rings with nominal diffraction angles of 40, 60, 90, 120 and 140, covering 22.4% of the 4p steradians around the sample [68]. The RPRs were mounted on cadmium-wrapped sample holders as described elsewhere [65]. The mounted samples were loaded on the HIPPO sample changer robot [69], and neutron diffraction data were collected at rotation angles of 0, 67.5 and 90 for 10 min per rotation, covering 51.7% of the 4p steradians [68]. The mea- sured diffraction patterns were analyzed using the Materials Analysis Using Diffraction (MAUD) soft- ware [70] via simultaneous Rietveld reﬁnement [71] of 132 neutron time-of-ﬂight diffraction patterns fol- lowing procedures described elsewhere [72]. An E- WIMV representation of 7.5 resolution was used to represent the orientation distribution function (ODF) in MAUD. No speciﬁc texture symmetry was assumed in the reﬁnement. Lastly, in order to gen- erate input for ELSC calculations of polycrystal elastic constants, the ODF was integrated in small, regularly spaced (every 10) volumes of orientation space u1; cos U; u2 ð Þ, where u1; U; u2 ð Þ are the Euler angles (Bunge convention), resulting in a set of 23,328 discrete orientations centered inside each volume, determining a 72918x18 partition of u1; cos U; u2 in the intervals ½0; 2p, ½0; 1 and ½0; p=2, respectively, with associated weights corresponding to the ODF integrals inside each volume. The ELSC model is a mean-ﬁeld approximation based on 1-point statistics, in which each grain is treated as an ellipsoidal elastic inhomogeneity embedded in an effective homogenized elastic med- ium. The ellipsoidal shape represents the average morphology of the grains. The inhomogeneity char- acter derives from the difference in elastic properties of each individual grain and the effective medium. The inhomogeneity and the medium will generally have fully anisotropic properties, deriving from the intrinsic single-crystal anisotropy, crystallographic texture and/or non-equiaxed morphology of the grains. 10059 10059 J Mater Sci (2021) 56:10053–10073 sample need to be oriented, for example, at 45 with respect to bz. sample need to be oriented, for example, at 45 with respect to bz. polycrystals, based on the knowledge of the proper- ties, morphology and orientation distribution of the constituent single-crystal grains. The ELSC method was proposed independently by Hershey [37] and generalized by Hill [73]. Here, we use the most recent ELSC numerical implementation by Tome´ and Lebensohn [74]. In the context of the SC theory, the polycrystal is represented by a set of weighted crystal orientations. The orientations represent grains, and the weights represent volume fractions. The set of orientations and weights are chosen to reproduce the crystallographic texture of the material. In this regard, the ELSC model utilizes the whole texture information contained in the full ODF measured by ND, instead of reduced representations, e.g., orien- tation distribution coefﬁcients used by Foster et al. [30] for cubic materials, or texture coefﬁcients for lower-symmetry crystals (e.g., [75] for hexagonal materials). Neutron diffraction The effective medium represents the average environment surrounding each grain. Using the equivalent Eshelby’s inclusion method [76] to solve each heterogeneity problem, the ELSC estimate for the polycrystal’s elastic stiffness (in tensorial nota- tion) is given by the expression: cijkl ¼ cg ijpq Ag pqkl D E ð14Þ ð14Þ where cijkl and cg ijkl are the elastic stiffness tensors of the polycrystal and each individual grain, the sym- bol \ . [ indicates weighted average over the set of grains representing the aggregate, and Ag ijkl is the localization tensor associated with each grain (g), which relates the strain applied to the polycrystal and the local strain in the grains, i.e., eg ij ¼ Ag ijkl ekl and is deﬁned by: Results and discussion Figure 3 Compressive and shear elastic constants of a 99.95% pure tungsten RPR as determined by 2-DoF and 9-DoF ﬁts using RUS. In order to determine the errors associated with an isotropic ﬁt (2 DoF) of an RUS spectrum for an elas- tically isotropic sample, a 99.95% pure polycrystalline tungsten RPR was measured using RUS and the elastic constants were determined using both iso- tropic (2 DoF) and orthorhombic (9 DoF) ﬁts. RPRs, and a microstructure that is homogeneous with a correctly modeled symmetry [24]. The slight devi- ations in the 9-DoF elastic constants, as well as the slightly smaller errors for the 9=DoF ﬁt in compar- ison with the 2-DoF ﬁt, are attributed to the avail- ability of more free parameters in the 9-DoF ﬁt minimization scheme. Given that the 9-DoF ﬁt is in such close agreement with the isotropic ﬁt, this demonstrates that the isotropic assumption is correct for this material. RPRs, and a microstructure that is homogeneous with a correctly modeled symmetry [24]. The slight devi- ations in the 9-DoF elastic constants, as well as the slightly smaller errors for the 9=DoF ﬁt in compar- ison with the 2-DoF ﬁt, are attributed to the avail- ability of more free parameters in the 9-DoF ﬁt minimization scheme. Given that the 9-DoF ﬁt is in such close agreement with the isotropic ﬁt, this demonstrates that the isotropic assumption is correct for this material. Tungsten was chosen because its elastic properties are inherently isotropic (A = 1.004 for single-crystal tungsten) [78]. This guarantees isotropic elastic properties irrespective of the polycrystal’s texture, because the elastic contributions of each grain are independently isotropic. RPRs of tungsten were machined using skim EDM with dimensions of 8.4 ± 0.0012 mm 9 9.2 ± 0.0015 mm 9 13.5 ± 0.001 mm resulting in a density of 19.135 ± 0.006 g cm-3, 99.2% that of the 19.3 gcm-3 nominal density [56]. Figure 4 displays the results of ﬁtting the RUS spectrum obtained from an AA-1100-O RPR using an isotropic ﬁt (2 DoF) and a hexagonal symmetry (5 DoF) for different orientations. For 2-DoF, the RMS error is 0.85%. This error is nearly ten times higher than for the isotropic ﬁt of the tungsten RPR. Elastic self-consistent model Self-consistent (SC) homogenization methods are used to estimate the mechanical response behavior of 10060 J Mater Sci (2021) 56:10053–10073 2 DoF 9 DoF 508 509 510 511 512 2 DoF 9 DoF 156.6 156.9 157.2 157.5 157.8 Compressive Elastic Constants (GPa) c11 c22 c33 Shear Elastic Constants (GPa) Fit c44 c55 c66 Figure 3 Compressive and shear elastic constants of a 99.95% pure tungsten RPR as determined by 2-DoF and 9-DoF ﬁts using RUS. 2 DoF 9 DoF 508 509 510 511 512 2 DoF 9 DoF 156.6 156.9 157.2 157.5 157.8 Compressive Elastic Constants (GPa) c11 c22 c33 Shear Elastic Constants (GPa) Fit c44 c55 c66 Ag ijkl ¼ cg þ ~c ð Þ1 ijpq c þ ~c ð Þpqkl ð15Þ ð15Þ where ~cijkl is the interaction tensor that gives the local deviations in stress in terms of the local deviations in stresses, i.e., rg ij rij ¼ ~cijkl eg kl ekl , and is given by: ~cijkl ¼ cijmn I S ð Þmnpq S1 pqkl ð16Þ ð16Þ where Sijkl is the Eshelby tensor [77], a function of cijkl and the shape of the ellipsoid representing the mor- phology of the grains [76]. Given these dependencies, Eq. (14) is a ﬁx-point equation that allows obtaining cijkl iteratively. j Note that Eq. (14) involves two weighted averages of the elastic moduli of each grain to give the poly- crystal’s stiffness tensor. The ﬁrst set of weights are the (tensorial) factors given by the localization ten- sors (Eq. 15), which depend on the relative stiffness and interaction between each grain and the effective medium representing the polycrystal. The second set of weights are the normalized values of the ODF integrals inside each of 23,328 volumes in orientation space, which provide the dependence with the polycrystal’s texture. Results and discussion The increase in the RMS for the 2-DoF ﬁt of the AA-1100 is not due to lack of accuracy in the density or geometry of these samples, as they are very similar to those of the tungsten RPRs. The elastic constants determined by these two ﬁt- ting methodologies, shown in Fig. 3, are identical (within error * 0.05%). These results show that a root-mean-square (RMS) error of 0.1% or lower can be achieved in RUS measurements if the sample geometry and microstructure are representative of the ﬁt used, i.e., sharp 90 corners and ﬂat faces for 10061 10061 J Mater Sci (2021) 56:10053–10073 Figure 4 a Bulk modulus compared to literature value [78] and b RMS error using different symmetry and orientation ﬁts for an AA-1100-O sample with c\|\|bz. values from the literature for polycrystalline alu- minum [79]. The complete determination of the elastic constants cij is shown in Table 1 for the 2- and 5-DoF ﬁts performed in the different orientations. It could also be argued that the lower RMS obtained with 5-DoF ﬁts is an artifact of a higher number of ﬁtting parameters used as compared to the 2-DoF ﬁt. However, the results for h\|\|a and h\|\|b indicate otherwise. There are cases when the true symmetry of the material is unknown a priori. The elastic constants can be extracted using a ﬁtting procedure with an even lower symmetry, such as an orthorhombic ﬁt (9 DoF) for which c11, c22, c33, c23, c13, c12, c44, c55 and c66 are independent. If the material truly has transversely isotropic texture, then the 9-DoF ﬁt should yield identical results (within errors) to the 5-DoF ﬁt. RMS error is expected to be slightly less for a 9-DoF ﬁt because there are more free parameters available in the minimization scheme, analogously to the tung- sten case. Figure 5 compares the results obtained using 5-DoF and 9-DoF ﬁts for the shear constants (c44, c55 and c66) of an AA-1100-O sample with c\|\|bz. The determined value of c66 is identical to the 5-DoF and 9-DoF ﬁts, and the values of c44 and c55 are within the error. More importantly, the results shown in Fig. 5 conﬁrm the assumption of transverse iso- tropy for the extruded material, without the need of determining the material’s symmetry or its orienta- tion a priori. Note that the level of anisotropy in the shear constants is clearly outside the error of deter- mination around 6%. Figure 4 a Bulk modulus compared to literature value [78] and b RMS error using different symmetry and orientation ﬁts for an AA-1100-O sample with c\|\|bz. Figure 4 a Bulk modulus compared to literature value [78] and b RMS error using different symmetry and orientation ﬁts for an AA-1100-O sample with c\|\|bz. The increase in RMS error indicates that the use of the isotropic ﬁt to analyze the RUS spectra of extru- ded AA-1100-O is not correct. Figure 6 RUS determination of elastic constants of AA-1100-O RPRs vs. physical orientation relative to texture axis, where all ﬁts were performed with h\|\|bz. results performed on two samples (3 and 5) for val- idation purposes, particularly the anisotropy ratios. From Fig. 7a, it can be seen that the cij determined by pulse echo are slightly lower than those determined All elastic constants/moduli are in GPA Fit Parameter 2-DoF 5-DoF Fit 5-DoF Fit 5-DoF Fit (GPA) Fit Assuming h\|\|a Assuming h\|\|b Assuming h\|\|c c11 100.0 109.4 99.8 111 c33 100.0 114.7 107.6 109 c13 47.4 61.6 53.1 59.6 c44 26.3 27 27.1 27.4 c66 26.3 26.1 26.7 25.9 B 64.9 77 67.8 76.4 RMS error (%) 0.85 0.67 0.62 0.11 Figure 5 Comparison of 9-DoF ﬁt vs. 5-DoF ﬁt shear elastic constants with c\|\|h using RUS for an AA-1100 sample machined with c\|\|bz. Table 1 Elastic constants and RMS error for an AA-1100-O sample machined c\|\|bz as determined by ﬁts with h\|\|a, h\|\|b and h\|\|c (best ﬁt). All elastic constants/moduli are in GPA Fit Parameter 2-DoF 5-DoF Fit 5-DoF Fit 5-DoF Fit (GPA) Fit Assuming h\|\|a Assuming h\|\|b Assuming h\|\|c c11 100.0 109.4 99.8 111 c33 100.0 114.7 107.6 109 c13 47.4 61.6 53.1 59.6 c44 26.3 27 27.1 27.4 c66 26.3 26.1 26.7 25.9 B 64.9 77 67.8 76.4 RMS error (%) 0.85 0.67 0.62 0.11 Figure 5 Comparison of 9-DoF ﬁt vs. 5-DoF ﬁt shear elastic constants with c\|\|h using RUS for an AA-1100 sample machined with c\|\|bz. Figure 5 Comparison of 9-DoF ﬁt vs. 5-DoF ﬁt shear elastic constants with c\|\|h using RUS for an AA-1100 sample machined with c\|\|bz. ﬁve independent constants, but both dilatational constants are shown in Fig. 6 for comparison purposes. In order to obtain more statistical conﬁdence in the anisotropy inferred from the results shown above, thirteen additional AA-1100-O samples were charac- terized, which were machined in the same fashion from the same rod and with identical thermal histo- ries as described before. The RUS spectra were col- lected for each of the RPRs and were used to perform hexagonal (5 DoF) ﬁts with h\|\|bz for each of the samples. The elastic constants for these samples are displayed in Fig. 7, along with the anisotropy ratios of the compressive and shear elastic constants. The anisotropy in the shear elastic constants (5.7% ± 0.5%) is consistently larger than the aniso- tropy in dilatational elastic constants (2.4% ± 0.6%) and larger than the anisotropy in compressive elastic constants (1.5% ± 0.5%) as determined by RUS. These ﬁndings are in close agreement with the PE Figure 6 RUS determination of elastic constants of AA-1100-O RPRs vs. physical orientation relative to texture axis, where all ﬁts were performed with h\|\|bz. For these AA-1100 samples, the effects of extrusion-induced texture cannot be ignored and are a signiﬁcant deviation from the assumption of the random polycrystalline orientation distribution. We also ﬁnd a 16% deviation from the literature value for bulk modulus B [79]. Thus, we analyze the same spectrum with a 5-DoF ﬁt (see Fig. 4) for a sample machined with c\|\|bz (see Fig. 1b). The 5-DoF ﬁt was then performed using different symmetry orientations, i.e., with h\|\|a, h\|\|b, and h\|\|c. Only the latter (h\|\|c, i.e., h\|\|bz) ﬁt matches with the sample’s symmetry. The RMS error decreases dramatically when the correct symmetry is chosen (see Fig. 4a). It is remarkable that although the RMS error of the 2-DoF ﬁt is 0.85%, the differences in the values obtained for B are as large as 16% (see Fig. 4b). This highlights the importance of choosing the correct symmetry. When ﬁtting with the correct symmetry, the values for B are consistent with the RUS is capable of determining the degree of elastic anisotropy for samples that were machined regard- less of the orientations with respect to the rod. The compressive, dilatational and shear elastic constants for samples machined in different orientations are shown in Fig. 6. Henceforth, all ﬁts were performed using a 5-DoF ﬁt with h\|\|bz. Although there are small variations in the elastic constants from sample to sample, this is likely due to handling and small dif- ferences in machining quality. The RMS errors of the ﬁts are all low, about 0.1%. The general relationships between the elastic constants are the same for each sample: c11 [ c33, c13 [ c12, and c44 [ c66. These sam- ples have much larger anisotropy in the shear con- stants than in the compressive constants. Note that c12 is set by the values of the other constants (see Eq. 9) since hexagonal symmetry is associated with J Mater Sci (2021) 56:10053–10073 Table 1 Elastic constants and RMS error for an AA-1100-O sample machined c\|\|bz as determined by ﬁts with h\|\|a, h\|\|b and h\|\|c (best ﬁt). J Mater Sci (2021) 56:10053–10073 To further investigate the origin of the elastic ani- sotropy, we performed neutron measurements to determine the texture and calculated the elastic ani- sotropy using self-consistent model using those neutron textures. Figure 8 shows the resulting pole ﬁgures and illustrations of the samples machined with the extrusion direction parallel to the c (Fig. 8a and b) and a (Fig. 8c and d). As shown in the pole ﬁgures, both samples have similar MRD values, indicating that, other than the rotation, texture is identical for the two samples. However, the plane of isotropy for the sample shown in Fig. 8a is clearly the a-b plane, while the plane of isotropy for the sample shown in Fig. 8c is the b-c plane, both of which are consistent with RUS and PE results. It is worth noting that there are very small deviations from transverse isotropy; e.g., the light blue ‘‘ring’’ in the top 111 pole ﬁgure is not of constant density around the ring, and the vertical band in the bottom 200 pole ﬁgure is not vertical. The determination of the degree of elastic aniso- tropy is paramount toward understanding the mechanical properties of the material. In this partic- ular case, with extruded AA-1100-O, anisotropy in the shear elastic constants is nearly 6% (see Fig. 7a). The engineering constants can be calculated from the elastic constants by using Eqs. 10–13. For these sam- ples, the Young’s moduli and Poisson ratios vary by 3% and 11%, respectively. As mentioned in ‘‘Neutron diffraction’’ section, the measured ODFs of samples c\|\|bz and a\|\|bz were integrated in small regions of orientation space to give a set of 23,328 orientations. These sets of indi- vidual orientations representing the textures of sam- ples c\|\|bz and a\|\|bz were used as input of ELSC calculations. J Mater Sci (2021) 56:10053–10073 J Mater Sci (2021) 56:10053–10073 their determination but in different levels of aniso- tropy among the samples. by the RUS technique. It is possible that this is due to the interaction of the propagating wave with the boundaries of the sample, which can guide and slow the wave, resulting in lower values of cij. We also see small variations in the elastic constants from sample to sample as expected due to different aspects such as sample inhomogeneity, handling and small differ- ences in machining quality. The variation is relatively bigger for the compression constants as the aniso- tropy is smaller. The difference in density is one order of magnitude smaller than the variations observed in cij values from sample to sample, thus not the origin of sample-to-sample variation found in cij. We did observe a non-systematic change in elastic constant obtained by RUS before and after sound velocity measurements, of the same order of the dispersion between samples. Nevertheless, the degree of anisotropy remains practically unchanged. by the RUS technique. It is possible that this is due to the interaction of the propagating wave with the boundaries of the sample, which can guide and slow the wave, resulting in lower values of cij. We also see small variations in the elastic constants from sample to sample as expected due to different aspects such as sample inhomogeneity, handling and small differ- ences in machining quality. The variation is relatively bigger for the compression constants as the aniso- tropy is smaller. The difference in density is one order of magnitude smaller than the variations observed in cij values from sample to sample, thus not the origin of sample-to-sample variation found in cij. We did observe a non-systematic change in elastic constant obtained by RUS before and after sound velocity measurements, of the same order of the dispersion between samples. Nevertheless, the degree of anisotropy remains practically unchanged. The determination of the degree of elastic aniso- tropy is paramount toward understanding the mechanical properties of the material. In this partic- ular case, with extruded AA-1100-O, anisotropy in the shear elastic constants is nearly 6% (see Fig. 7a). The engineering constants can be calculated from the elastic constants by using Eqs. 10–13. For these sam- ples, the Young’s moduli and Poisson ratios vary by 3% and 11%, respectively. 10063 J Mater Sci (2021) 56:10053–10073 The other adopted input parameters were the values of Al single-crystal elastic constants cAlSX 11 ¼ 107 GPa, cAlSX 12 ¼ 61 GPa and cAlSX 44 ¼ 28 GPa obtained by averaging ﬁve different mea- surements at room temperature reported in [78], and a grain’s ellipsoidal shape (1:1:5) along x, y and z, consistent with the morphology of grains extruded in bz: (Note that the reported results are fairly insensitive to the actual ratio between the long axis and the short axes of the ellipsoid.) Another common measure of anisotropy was pro- posed by Thomsen [80], given by these parameters: e ¼ c11 c33 2c33 c ¼ c66 c44 2c44 d ¼ c13 þ c44 ð Þ2 c33 c44 ð Þ2 2c33 c33 c44 ð Þ ð17Þ ð17Þ The ﬁrst two anisotropy parameters (e and c) are the normalized difference of the compressive and shear elastic constants. The parameter d reﬂects the near-vertical compression wave anisotropy and does not include c11 (the horizontal velocity) [80]. For weak anisotropy, d can be further reduced to dweak = (c13- (c33-2c44))/c33, so d can also be seen as the off-diag- onal or dilatational constants anisotropy factor. Interestingly, when the d is plotted against e and c, we ﬁnd a clear correlation between them with d = 0.036 ? 2.88e and d = - 0.01 ? 2.17 c in Fig. 7b. This suggests that the differences in cij between samples are not due to random variation or error in Figure 9 shows the ELSC predictions of nine polycrystal’s elastic stiffness components: c11; c22; c33;c12; c13; c23 and c44; c55; c66 for samples c\|\|bz and a\|\|bz. Note that symmetry of cijkl was not imposed but arose naturally from the symmetry of the input textures. It is observed that: a) the predicted polycrystal elastic constants are very close to trans- versely isotropic symmetry (or ‘‘axial symmetry’’ or ‘‘hexagonal symmetry’’). J Mater Sci (2021) 56:10053–10073 For example, in the case of c\|\|bz sample, c11 ﬃc22, c13 ﬃc13, c44 ﬃc55 and c12 ﬃ 10064 J Mater Sci (2021) 56:10053–10073 0 1 2 3 4 5 6 7 8 9 10 11 12 13 105 108 111 114 117 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 25 26 27 28 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0.98 1 1.02 1.04 1.06 1.08 (a) Compressive Constants (GPa) Sample c11 - RUS c11 - PE c33 - RUS c33 - PE Thomsen's anisotropy parameter δ Anisotropy parameter δ Shear Constants (GPa) c44 - RUS c44 - PE c66 - RUS c66 - PE Anisotropy Ratio c11/c33 - RUS c11/c33 - PE c44/c66 - RUS c44/c66 - PE 0 1 2 3 4 5 6 7 8 9 10 11 12 13 105 108 111 114 117 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 25 26 27 28 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0.98 1 1.02 1.04 1.06 1.08 (a) (b) Compressive Constants (GPa) Sample c11 - RUS c11 - PE c33 - RUS c33 - PE Thomsen's anisotropy parameter δ Anisotropy parameter δ Shear Constants (GPa) c44 - RUS c44 - PE c66 - RUS c66 - PE Anisotropy Ratio c11/c33 - RUS c11/c33 - PE c44/c66 - RUS c44/c66 - PE Thomsen's 0 1 2 3 4 5 6 7 8 9 10 11 12 13 (b) Sample (b) S ample (b) 10065 J Mater Sci (2021) 56:10053–10073 bFigure 7 a Elastic constants and anisotropy ratios of several AA- 1100 RPRs as determined by RUS and PE. Error bars are the same size as the symbols, b Thomsen’s anisotropy parameter d as a function of e and c. bFigure 7 a Elastic constants and anisotropy ratios of several AA- 1100 RPRs as determined by RUS and PE. Error bars are the same size as the symbols, b Thomsen’s anisotropy parameter d as a function of e and c. becoming the direction of the symmetry axis, and consequently c11 6¼ c22 ﬃc33, c23 6¼ c12 ﬃc13, c44 6¼ c55 ﬃc66 and c23 ﬃc33 2c44. These results, obtained from textures of samples cut from the same rod but independently measured, show consistency between the texture measurements and modeling methodology. c11 2c66 within a tolerance better than 0.1%; b) the difference between the c\|\|bz and a\|\|bz is essentially a switch between the sample directions, z and x, with x c11 2c66 within a tolerance better than 0.1%; b) the difference between the c\|\|bz and a\|\|bz is essentially a switch between the sample directions, z and x, with x Figure 10 shows the comparisons between the polycrystal’s elastic constants measured with RUS Figure 8 a Neutron diffraction pole ﬁgures for an aluminum 1100 RPR whose extrusion direction is parallel to ‘‘c’’ (c\|\|bz) as illustrated in (b), and c neutron diffraction pole ﬁgures for an aluminum 1100 RPR whose extrusion direction is parallel to ‘‘a’’ (a\|\|bz) as illustrated in (d). Figure 8 a Neutron diffraction pole ﬁgures for an aluminum 1100 RPR whose extrusion direction is parallel to ‘‘c’’ (c\|\|bz) as illustrated in (b), and c neutron diffraction pole ﬁgures for an aluminum 1100 RPR whose extrusion direction is parallel to ‘‘a’’ (a\|\|bz) as illustrated in (d). 109.0 109.5 110.0 110.5 111.0 111.5 112.0 25.0 25.5 26.0 26.5 27.0 58.5 59.0 59.5 60.0 60.5 c\|\|z, ELSC a\|\|z, ELSC C33 C22 C11 C11 C22 C33 Cij [GPa] C66 C55 C44 C66 C55 C44 C12 C13 C23 C13 C23 C12 Figure 9 ELSC predictions of polycrystal’s elastic constants with input from ND textures for samples c\|\|bz and a\|\|bz. 10066 J Mater Sci (2021) 56:10053–10073 and predicted with ELSC with input from ND tex- tures for samples c\|\|bz and a\|\|bz, respectively. Note that ranges of all vertical axes are 4 GPa, for a fair visual comparison of the results. J Mater Sci (2021) 56:10053–10073 It is observed that the predicted anisotropy is qualitatively consistent with the RUS measurements, e.g., in the case of sample c\|\|bz: c33\c11 ﬃc22, c12\c13 ﬃc23, and c66\c44 ﬃc55, although quantitative, the predictions and measurement are off, in some of the worst cases, by several percent. The quantitative differences between calculation and experiments may be due to systematic errors in the texture and intrinsic to the RUS analysis, namely, the measured texture by neu- trons is not perfectly transverse isotropic as assumed (see Fig. 8), the single crystal elastic constants of AA- 1100-O are not the ones found in a single crystal extracted from tables, and/or model shortcomings. Elucidation of the ultimate causes of quantitative disagreement between experiments and predictions is currently the subject of investigation, including the 108 109 110 111 112 (a) (b) 24 25 26 27 28 56 57 58 59 60 RUS ELSC C11=C22 C33 C11 C22 C33 Cij [GPa] C44=C55 C66 C66 C55 C44 C13=C23 C12 C13 C23 C12 108 109 110 111 112 24 25 26 27 28 56 57 58 59 60 RUS ELSC C22=C33 C11 C22 C33 C11 Cij [GPa] C55=C66 C44 C44 C66 C55 C12=C13 C23 C12 C13 C23 Figure 10 a Elastic constants for a polycrystalline sample c\|\|bz measured with RUS and predicted with ELSC with input from ND textures. b Elastic constants for a polycrystalline sample a\|\|bz measured with RUS and predicted with ELSC with input from ND textures. between calculation and experiments may be due to systematic errors in the texture and intrinsic to the RUS analysis, namely, the measured texture by neu- trons is not perfectly transverse isotropic as assumed (see Fig. 8), the single crystal elastic constants of AA- 1100-O are not the ones found in a single crystal extracted from tables, and/or model shortcomings. Elucidation of the ultimate causes of quantitative disagreement between experiments and predictions is currently the subject of investigation, including the and predicted with ELSC with input from ND tex- tures for samples c\|\|bz and a\|\|bz, respectively. Note that ranges of all vertical axes are 4 GPa, for a fair visual comparison of the results. It is observed that the predicted anisotropy is qualitatively consistent with the RUS measurements, e.g., in the case of sample c\|\|bz: c33\c11 ﬃc22, c12\c13 ﬃc23, and c66\c44 ﬃc55, although quantitative, the predictions and measurement are off, in some of the worst cases, by several percent. J Mater Sci (2021) 56:10053–10073 The quantitative differences between calculation and experiments may be due to systematic errors in the texture and intrinsic to the RUS analysis, namely, the measured texture by neu- trons is not perfectly transverse isotropic as assumed (see Fig. 8), the single crystal elastic constants of AA- 1100-O are not the ones found in a single crystal extracted from tables, and/or model shortcomings. Elucidation of the ultimate causes of quantitative disagreement between experiments and predictions is currently the subject of investigation, including the 10067 J Mater Sci (2021) 56:10053–10073 study of materials with higher intrinsic single-crystal anisotropy. no cold working), which has transversely isotropic symmetry (ﬁve independent elastic constants) due to the texture induced by the extrusion process. The relative anisotropy of the compressive (c11 vs. c33) and shear (c44 vs. c66) elastic constants is 1.5% ± 0.5% and 5.7% ± 0.5%, respectively, where c33 and c66 are the elastic constants associated with the axis of symmetry of the texture. Because aluminum is an inherently low-anisotropy material whose Zener anisotropy factor is 1.22, other common alloys (steels, nickel-based superalloys, copper-based alloys, etc.) are expected to have larger anisotropic properties given the same texture. As mentioned before, aluminum has a relatively low Zener anisotropy factor of A = 1.22, meaning that its elastic properties are less sensitive to anisotropic changes due to texture than, say copper (A = 3.21), or 304 stainless steel (A = 3.77) [44] and many other common materials with larger A factors, for which the degree of anisotropy due to texture will be much larger. Indeed, using the same texture measured for AA-1100, we calculated the polycrystal elastic con- stants that would result from Cu and SS samples with identical texture, as shown in Fig. 11, using a nor- malization based on the corresponding isotropic elastic constants in the case of random texture, for each material. Clearly, the same texture has larger effect on the elastic anisotropy as the A increases. The changes in compression constants are smaller than those found in shear. We ﬁnd an overall decrease in the average of the compression constant with higher A. For shear, the anisotropy is larger and there is a decrease in c66 clearly seen for Cu and SS. J Mater Sci (2021) 56:10053–10073 g The necessity of accounting for the anisotropy resulting from the extrusion process has been demonstrated by comparing the RUS results from AA-1100-O samples ﬁt using the isotropic assump- tion (2 DoF), the transverse isotropic (5 DoF) and orthotropic (9 DoF) ﬁts. The error associated with describing extruded aluminum with an isotropic ﬁt is supported by the extremely low-error, high-accuracy RUS results of the isotropic ﬁt in 99.95% pure tung- sten, which has a Zener anisotropy factor of unity and is therefore isotropic regardless of texture. The ability for RUS to determine the orientation of the texture axis has also been demonstrated by analyzing the results with a lower symmetry (9 DoF) and changing the orientation of the ﬁt with respect of extrusion orientation (5 DoF). The elastic constants and the degree of elastic anisotropy as determined Appendix The relationship between the phase velocities of ultrasonic plane waves and the elastic constants of the material can be determined by solving the eigenvalue problem associated with the Christoffel equation in Eq. 18, where q is the density, v is the elastic wave phase velocity, dij is the Kronecker delta, Cij = cijklnjnl is the Christoffel tensor, and nj are the directional cosines of the elastic wave propagation direction for a given reference axis [81–83]. For iso- tropic materials (two independent elastic constants, see Eq. 3 where c12 = c11–2c44), the Christoffel tensor reduces to the relations in Eq. 19: Summary and conclusions In this study, we use resonant ultrasound spec- troscopy (RUS) to nondestructively determine the entire elastic tensor in extruded AA-1100-O (com- mercially pure aluminum in its annealed state with Figure 11 Normalized elastic constants (vs. randomly distributed elastic constant) of AA-1100, Cu and SS using the same experimental texture. Figure 11 Normalized elastic constants (vs. randomly distributed elastic constant) of AA-1100, Cu and SS using the same experimental texture. 10068 J Mater Sci (2021) 56:10053–10073 using RUS have been conﬁrmed by direct sound velocity measurements using the ultrasonic pulse- echo technique. The elastic constants were then used to calculate the engineering constants, whereby the Young’s moduli and Poisson ratios parallel vs. per- pendicular to the extrusion direction in these samples differ by 3% and 11%, respectively. Using the experimental texture obtained by neutron diffraction, elastic self-consistent micromechanical simulations show good agreement in the level of anisotropy and small disagreement in the absolute values that we assign to material changes produced by handling the soft-annealed AA-1000 samples. Using the same texture and calculation method, we show that the texture will have much larger effect for materials with larger Zener parameter, resulting even in low- ering of the elastic constants. 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/licen ses/by/4.0/. Acknowledgements Research presented in this article was supported by the Laboratory Directed Research and Development program of Los Alamos National Laboratory under project 20180250ER. Work at the National High Magnetic Field Laboratory at LANL, was also sup- ported by the National Science Foundation through NSF/DMR-1644779 and the state of Florida (F.F.B., J.B.). Work by B.T.S. was supported by LANL Ofﬁce of Experimental Science Dynamic Materials Proper- ties program (Campaign 2). The work has beneﬁtted from the use of the Los Alamos Neutron Science Center (LANSCE) at LANL. Los Alamos National Laboratory is operated by Triad National Security, LLC, for the National Nuclear Security Administra- tion of the U.S. Department of Energy under contract number 89233218NCA000001. Cij qv2dij ¼ 0 Cij ¼ C11 C12 C13 C21 C22 C23 C31 C32 C33 ð18Þ ð18Þ C11 ¼ c11n2 1 þ c44n2 2 þ c44n2 3 C22 ¼ c44n2 1 þ c11n2 2 þ c44n2 3 C33 ¼ c44n2 1 þ c44n2 2 þ c11n2 3 C12 ¼ n1n2 c12 þ c44 ð Þ C23 ¼ n2n3 c12 þ c44 ð Þ C13 ¼ n1n3 c12 þ c44 ð Þ ð19Þ ð19Þ Compliance with ethical standards J Mater Sci (2021) 56:10053–10073 J Mater Sci (2021) 56:10053–10073 c11 sin2 h þ c66 cos2 h qv2 c12 þ c66 ð Þ sin h cos h 0 c12 þ c66 ð Þ sin h cos h c66 sin2 h þ c11 cos2 h qv2 0 0 0 c44 qv2 2 64 3 75 ux uy uz 2 64 3 75 ¼ 0 0 0 2 64 3 75 ð24Þ 10069 c11 sin2 h þ c66 cos2 h qv2 c12 þ c66 ð Þ sin h cos h 0 c12 þ c66 ð Þ sin h cos h c66 sin2 h þ c11 cos2 h qv2 0 0 0 c44 qv2 2 64 3 75 ux uy uz 2 64 3 75 ¼ 0 0 0 2 64 3 75 ð24Þ c44 qv2 2 c11 qv2 ¼ 0 ð21Þ ð21Þ where ui is the polarization vector which represents the atomic displacement directions. The roots of the above relationship are the eigenvalues of the Christoffel tensor which relate the transverse (vS) and longitudinal (vL) phase velocities to the elastic con- stants of an isotropic solid, as shown in Eq. 22: where ui is the polarization vector which represents the atomic displacement directions. The roots of the above relationship are the eigenvalues of the Christoffel tensor which relate the transverse (vS) and longitudinal (vL) phase velocities to the elastic con- stants of an isotropic solid, as shown in Eq. 22: ð24Þ c11 sin2 h þ c44 cos2 h qv2 0 c13 þ c44 ð Þ sin h cos h 0 c66 sin2 h þ c44 cos2 h qv2 0 c13 þ c44 ð Þ sin h cos h 0 c44 sin2 h þ c33 cos2 h qv2 2 64 3 75 ux uy uz 2 64 3 75 ¼ 0 0 0 2 64 3 75 ð25Þ c11 sin2 h þ c44 cos2 h qv2 0 c13 þ c44 ð Þ sin h cos h 0 c66 sin2 h þ c44 cos2 h qv2 0 c13 þ c44 ð Þ sin h cos h 0 c44 sin2 h þ c33 cos2 h qv2 2 64 3 75 ux uy uz 2 64 3 75 ¼ 0 0 0 2 64 3 75 ð25Þ ð25Þ The ultrasonic phase velocities relate to the elastic constants of a transversely isotropic material by Eq. J Mater Sci (2021) 56:10053–10073 26, where vT,q, vT and vL are the quasi-transverse, pure transverse and longitudinal velocities, and h is the angle between the propagation direction and the z-axis of symmetry (i.e., h = 0 for waves propagating along the z-axis). c44 ¼ qv2 T c11 ¼ qv2 L ð22Þ c44 ¼ qv2 T c11 ¼ qv2 L ð22Þ Note that because there is no orientation depen- dence in isotropic materials, the entire elastic tensor can be determined from a single pulse-echo experi- ment. For transversely isotropic materials (ﬁve inde- pendent elastic constants, see Eq. 9), however, the Christoffel tensor is described by the relations in Eq. 23. In this analysis, the plane of isotropy has been assigned to the x–y plane, and the ‘‘z’’ direction is parallel to the textured axis. vL ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ c11 sin2 h þ c33 cos2 h þ c44 þ ﬃﬃﬃﬃﬃ M p 2q s vT;q ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ c11 sin2 h þ c33 cos2 h þ c44 ﬃﬃﬃﬃﬃ M p 2q s vT ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ c66 sin2 h þ c44 cos2 h q s M ¼ c11 c44 ð Þ sin2 h þ c33 c44 ð Þ cos2 h 2 þ c13 þ c44 ð Þ2sin2 2h : ð26Þ vL ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ c11 sin2 h þ c33 cos2 h þ c44 þ ﬃﬃﬃﬃﬃ M p 2q s vT;q ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ c11 sin2 h þ c33 cos2 h þ c44 ﬃﬃﬃﬃﬃ M p 2q s vT ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ c66 sin2 h þ c44 cos2 h q s M ¼ c11 c44 ð Þ sin2 h þ c33 c44 ð Þ cos2 h 2 þ c13 þ c44 ð Þ2sin2 2h : ð26Þ ð26Þ C11 ¼ c11n2 1 þ c66n2 2 þ c44n2 3 C22 ¼ c66n2 1 þ c11n2 2 þ c44n2 3 C33 ¼ c44n2 1 þ c44n2 2 þ c33n2 3 C12 ¼ n1n2 c12 þ c66 ð Þ C23 ¼ n2n3 c13 þ c44 ð Þ C13 ¼ n1n3 c13 þ c44 ð Þ ð23Þ M ¼ c11 c44 ð Þ sin2 h þ c33 c44 ð Þ cos2 h 2 þ c13 þ c44 ð Þ2sin2 2h ð23Þ The relationship between sound velocities and sample orientations for c13 is given in Eq. 27 for an experiment performed at 45 from the texture axis. Compliance with ethical standards Since changing the orientation in which the elastic wave velocity measurement is performed in an iso- tropic material yields identical results, we arbitrarily assign the z-axis as the direction of elastic wave propagation, i.e., nx = ny = 0 and nz = 1. It follows that: Conﬂict of interest The authors declare no com- peting interest. Open Access This article is licensed under a Crea- tive 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 Crea- tive Commons licence, and indicate if changes were made. The images or other third party material in this c44 qv2 0 0 0 c44 qv2 0 0 0 c11 qv2 2 4 3 5 ux uy uz 2 4 3 5 ¼ 0 0 0 2 4 3 5 ð20Þ ð20Þ and J Mater Sci (2021) 56:10053–10073 Samples were not machined in such off-angle orien- tations in this work; therefore, pulse echo was unable to obtain information related to the values of c13 for the AA-1100 samples in this study. Under these symmetry conditions, the direction of wave propagation is now relevant. Following the same methodology as before, we now consider a plane wave propagating in the x–y plane in a direc- tion h with respect to the z-axis. Therefore, n1 = sinh, n2 = cosh, n3 = 0, and the Christoffel equation becomes Eq. 24. Similarly, for a plane wave propa- gating in the x–z plane in a direction h with respect to the z-axis, n1 = sinh, n2 = 0, n3 = cosh, and the Christoffel equation becomes Eq. 25. c11 ¼ qv2 L h¼90 c33 ¼ qv2 L h¼0 c12 ¼ c11 2c66 c13 ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 4q2v4 L h¼452qv2 L h¼45 c11 þc33 þ2c44 ð Þþ c11 þc44 ð Þ c33 þc44 ð Þ q c44 c44 ¼ qv2 T h¼0 c66 ¼ qv2 T h¼90 ð27Þ 10070 J Mater Sci (2021) 56:10053–10073 Sci Eng A-Struct Mater Prop Microstruct Process 722:20–29. https://doi.org/10.1016/j.msea.2018.02.081 Sci Eng A-Struct Mater Prop Microstruct Process 722:20–29. https://doi.org/10.1016/j.msea.2018.02.081 [9] Chen JA, Yan W, Li W, Miao JA, Fan XH (2011) Texture evolution and its simulation of cold drawing copper wires produced by continuous casting. 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https://openalex.org/W4367314752	http://www.mjssm.me/clanci/MJSSM_March_2023_Bourogiannis.pdf	English	null	Reintegration Program for Professional Football Players after Grade 2 Hamstring Injuries	Montenegrin Journal of Sports Science and Medicine	2,023	cc-by	5,832	ORIGINAL SCIENTIFIC PAPER Abstract Hamstring strains are among the most common injuries in soccer, with an increased risk of recurrence. There are three stages in injuries: 1 Diagnosis, 2 Physiotherapy – Rehabilitation, 3 Reintegration. The present study inves- tigated the reintegration actions (return to play – RTP) for eight professional footballers (Super League 1) after hamstring strain. Eight professional soccer players who had hamstring injuries with Grade 2 strain were measured and evaluated. Two measurements were taken before and after injury. The first measurement involved maximum effort field tests of 30, 40, 50 and 60 meters in a straight line without a ball, using global positioning system (GPS) devices placed on the athletes for the interpretation and evaluation of their results. The second measurement aimed to evaluate the maximum strength of the eight players before and after their injury, using squats, bench presses and leg curl exercises on the machine. The statistical analysis included descriptive and inductive statistics (paired t-test). Analysis of the data collected showed that all soccer players had higher performance in the mea- surements taken after injury, both in speed and in maximal strength. In conclusion, following the appropriate rehabilitation and reintegration, the eight athletes were not affected by their injury and managed to return to competitive action (RTP) without recurrence and maximizing their performance. Keywords: Reintegration, injury, hamstrings, footballer, rehabilitation Cite this article: Bourogiannis, G., Hatzimanouil, D., Semanltianou, E., Georgiadis, I., & Sykaras, E. (2023) Reintegration Program for Professional Football Players after Grade 2 Hamstring Injuries. Montenegrin Journal of Sports Science and Medicine, 19 (1), 35–42. https://doi.org/10.26773/mjssm.230305 Reintegration Program for Professional Football Players after Grade 2 Hamstring Injuries Georgios Bourogiannis1, Dimitris Hatzimanouil1, Eleni Semanltianou1, Ioannis Geo Affiliations: 1Aristotle University Of Thessaloniki, Department of Physical Education & Sport Science Correspondence: G. Bourogiannis. Aristotle University of Thessaloniki, Department of Physical Educatio Thessaloniki. E-mail: giwrgosbouro@gmail.com Abstract Hamstring strains are among the most common injuries in soccer, with an increased three stages in injuries: 1 Diagnosis, 2 Physiotherapy – Rehabilitation, 3 Reintegratio tigated the reintegration actions (return to play – RTP) for eight professional footba hamstring strain. Eight professional soccer players who had hamstring injuries with G and evaluated. Two measurements were taken before and after injury. The first measu effort field tests of 30, 40, 50 and 60 meters in a straight line without a ball, using glob devices placed on the athletes for the interpretation and evaluation of their results aimed to evaluate the maximum strength of the eight players before and after their presses and leg curl exercises on the machine. The statistical analysis included descrip (paired t-test). Analysis of the data collected showed that all soccer players had high surements taken after injury, both in speed and in maximal strength. In conclusion rehabilitation and reintegration, the eight athletes were not affected by their injury competitive action (RTP) without recurrence and maximizing their performance. Keywords: Reintegration, injury, hamstrings, footballer, rehabilitation Cite this article: Bourogiannis, G., Hatzimanouil, D., Semanltianou, E., Georgiad Reintegration Program for Professional Football Players after Grade 2 Hamstring In of Sports Science and Medicine, 19 (1), 35–42. https://doi.org/10.26773/mjssm.2303 @MJSSMontenegro REINTEGRATION PROGRAM FOR PROFESSIONAL FOOTBALL PLAYERS http://mjssm.me/?sekcija=article&artid=250 Affiliations: 1Aristotle University Of Thessaloniki, Department of Physical Education & Sport Science ce: G. Bourogiannis. Aristotle University of Thessaloniki, Department of Physical Education & Sport Science, Thermi, 57001, -mail: giwrgosbouro@gmail.com Correspondence: G. Bourogiannis. Aristotle University of Thessaloniki, Department of Physical Education & Sport Scie Thessaloniki. E-mail: giwrgosbouro@gmail.com Received: 12 November 2022 \| Accepted after revision: 26 February 2023 \| Early access publication date: 1 March 2023 \| Final publication date: 15 March 2023 © 2023 by the author(s). License MSA, Podgorica, Montenegro. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY). Introduction majority of training and competitive actions in intermittent sports like football occur within 5-30m (Carling et al., 2016). They evaluated speed using a 30-meter sprint test with speed measured at 10 meters, 20 meters and 30 meters (Altmann et al., 2019). However, the speed at distances of 40, 50 and 60 meters has not been investigated. These distances 40,50,60 meters can occur in football firstly, in actions during the game by the fullbacks, who have to attack and return to their position back in defense in case the ball is lost, and secondly, Hamstring injuries are common in many sports including football (Kujala et al., 1997). Strain is the typical pattern of injury and results from excessive stretching of the muscle, often during sprinting or jumping (Slavotinek et al., 2010). More than 70% of football players injure their hamstrings during sprints (Skling et al., 2014). Failure to properly rehabilitate – reintegrate or an early return can lead to a relapse and a lost season (Mendez et al., 2022). It has been investigated that the Received: 12 November 2022 \| Accepted after revision: 26 February 2023 \| Early access publication date: 1 March 2023 \| Final publication date: 15 March 2023 © 2023 by the author(s). License MSA, Podgorica, Montenegro. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY). 35 DOI 10.26773/mjssm.230305 REINTEGRATION PROGRAM FOR PROFESSIONAL FOOTBALL PLAYERS \| G. BOUROGIANNIS ET AL. and one was a fullback. Before being injured, the players were subjected to the following tests: speed field test using GPS at 30, 40, 50 and 60 meters, and biceps unipedal flexions with maximum force of 1RM on the leg curl machine. Depending on the position they were competing in, the players had to cover specific high intensity meters in the speed field test (Bayer et al., 2018). At the beginning of stage 1 of the players’ injury, the team’s orthopedic doctor performed an MRI and ultrasound scans to accurately determine the extent of the injuries. They were evaluated and determined by the orthopedic doctor to be grade 2 contusions with muscle fiber tears. Introduction At the end of stage 2 and before the start of stage 3, which involves reintegration, the same diagnostic tests (MRI and ultrasound scans) were performed again to determine if there was muscle healing and if the athletes were ready to begin reintegration.ht when central defenders in static situations are advanced to score and have to run quickly back into defense in case of a counter attack by the opposing team. For the above reasons our players were also subjected to the 40,50 and 60 meters speed tests. (Diagnosis, which is stage 1, is performed on players to determine the severity of the injury and make a proper evaluation. This is usually done clinically with either magnetic resonance imaging (MRI) or ultrasound. This is very important as a first step to have an accurate knowledge of the extent of the injury and the length of time the player will be absent from active play (Schneider-Kolsky et al., 2006). Furthermore, MRI offers a detailed analysis of the injury and is the preferred method for elite athletes in an attempt to prevent re-injury for those players who may return to training prematurely (Mendiguchia J et al., 2011). The second stage, concerning physical therapies to be followed by football players, consists of proper management to relieve the pain and for the gradual healing of the torn muscle (Cohen – Bradley et al., 2007). With stage 2 the players should have fully gained full range of motion of their limb. With another diagnostic test to confirm that the muscle has healed they are ready for stage 3, rehabilitation (Askling et al., 2007). High intensity total distance and sprints are extremely important factors in the reintegration stage for returning football players to a better condition than they were before the injury (Mohr et al., 2003). Therefore, the amount of high intensity running combined with lower limb maximum strength training can be considered indicators of increased performance after a hamstring injury (Tol et al., 2014). h The initial tests were performed after preparation as s baseline measurement and a benchmark. The time period from injury to return to competitive action was the same for all players. You cannot know when a player will be injured. So the players had the same recovery time and were evaluated in the same time period after their injury. Introduction The results are comparable because there was no focus on the time between the initial measurement and the injury, for the reason that an injury cannot be predicted. h The eight professional football players had no previous injuries, so they were selected and not excluded from the study. 1 Midfielder: age 33, height 1.85, weight 78, BMI 22, 79. 2 Midfielder: age 29, height 1.82, weight 75, BMI 22, 64. 3 Midfielder: age 25, height 1.71, weight 65, BMI 22, 23. i 4 Striker: age 21, height 1.86, weight 77, BMI 22, 26. 5 Striker: age 29, height 1.88, weight 80, BMI 22, 63. 6 Winger: age 24, height 1.79, weight 75, BMI 23, 41. 7 Central defender: age 27, height 1.93, weight 80, BMI 21, 75. 8 Fullback : age 26, height 1.74, weight 67, BMI 22, 13. h t Therefore, with appropriate/correct rehabilitation and reintegration, professional footballers restore their high performance standards and maximize them by returning to better physical fitness than before (Sherry & Best, 2004). About the mechanisms, hamstring injuries occur in sport activities requiring sprinting, cutting movements, acceleration efforts and extreme stretch movements (Schache et al., 2012). However, within the hamstring muscular complex it is important to split the injury mechanisms into biceps femoris (BF), semimembranosus (SM), and semitendinosus (ST) muscles lesions. The hamstring muscular complex during the running biomechanics is active from the beginning of the mid-swing phase until the terminal stance phase (Drezner et al., 2003). During this period of time, the BF is the muscle that undergoes the most elongation, equal to approximately 12% of its rest length. In the same phase, the SM is the flexor muscle producing the most important strength peak and absorbing the most important parts of the power production (Schache et al., 2012). For these reasons, the BF and SM injuries are substantially different. In other words, the BF injury mechanism is mainly based on an overstretching event, while the SM injuries are mainly based on a strength/power overproduction (Askling et al.,2007). This led to the aim of the present study, which was the investigation of the reintegration actions (return to play – RTP) through an appropriate individual program and tests, for eight professional footballers (Super League 1) after grade 2 hamstring strain. Introduction Inclusion: The football players included in the study participated in all training sessions, had playing time in official matches and had no serious injuries in the previous season. Exclusion: The football players excluded from survey did not participate in all training sessions, had no playing time in official matches and had serious injuries in the previous season. Speed field test using GPS For the purposes of the test, the midfielders had to cover a total distance of 600 meters at very high intensity (VHI Drills / maximal intent), 3 sets of 30 meters – 3 sets of 40 meters – 3 sets of 50 meters – 4 sets of 60 meters. There was a break of 30- 40 seconds between the repeats and a 3-minute break between sets. The footballers competing in the attack had to cover a total distance of 720 meters at very high intensity (VHI Drills / maximal intent), 5 sets of 30 meters – 3 sets of 40 meters – 3 sets of 50 meters – 5 sets of 60 meters. There was a break of 30-40 seconds between the repeats and a 3-minute break between sets. The lateral striker had to cover a total distance of 720 meters at very high intensity (VHI Drills / maximal intent), 5 sets of 30 meters – 3 sets of 40 meters – 3 sets of 50 meters – 5 sets of 60 meters. There was a break of 30-40 seconds between the repeats and a 3-minute break between sets. The central defender had to cover a total distance of 540 meters at very high intensity (VHI Drills / maximal intent), 3 sets of 30 meters – 3 sets of 40 meters – 3 sets of 50 meters – 3 sets of 60 meters. There was a break of 30-40 seconds between the repeats and a 3-minute break REINTEGRATION PROGRAM FOR PROFESSIONAL FOOTBALL PLAYERS \| G. BOUROGIANNIS ET AL. Table 1 shows the distances (meters) covered by our football players during matches and the average high-intensity distance (speed over 18 km km/h) covered during a match at the professional level depending on the position of each player. We selected our study’s speed limits according to our team’s GPS data. between sets. The lateral defender had to cover a total distance of 720 meters at high intensity (VHI Drills / maximal intent), 5 sets of 30 meters – 3 sets of 40 meters – 3 sets of 50 meters – 5 sets of 60 meters. There was a break of 30-40 seconds between the repeats and a 3-minute break between sets. Table 1. High Intensity Distances (Meters) Covered by our football Players during Matches Table 1. High Intensity Distances (Meters) Covered by our football Players during Matches Player position Average – level of our professional team Central Defenders 430 meters Fullbacks 680 meters Central Midfielders 580 meters Wide Midfielders 740 meters Attackers 620 meters Average 630 meters Average – level of our professional team Maximum test measurement 1RMh attempts. All measurements were performed with a fixed body position, using the same resistance equipment, by the same trainer (Śliwowski et al., 2015). The football players underwent the prescribed 1RM maximum force measurement for one exercise in the training program, the leg curl exercise. Prior to the 1RM test all participants followed a standard warm-up routine of one set of 10 repetitions with approximately 50% of the sub maximal loads to follow, using the correct movement technique. To determine the sub maximal force loads, a progressive increase in kilograms was performed for each exercise until the football players were unable to complete a repetition with correct technique. A 3-5 minute break was given between sets. 1RM was achieved between 4-5 Reintegration stage (training process) During the 3rd stage of their injury, the reintegration stage, all eight players followed a program which lasted 11 days, and on the 12th day they started training with the rest of the team. The program, described in Table 2, represents a very high intensity progression during rehabilitation of a grade 2 hamstring strain injury, taking into account an average very high intensity game profile of 630 meters (Very High Intensity Distance). Table 2. Methodsh The study sample consisted of eight professional football players (Super League 1) who had suffered a grade 2 strain in the first half of the 2020-2021 season. Three were midfielders, two were strikers, one was a winger, one was a central defender DOI 10.26773/mjssm.230305 36 Statistical Analysis whether there was a difference between the maximum right hamstring strength before and after the strain. Kolmogorov-Smirnov and Shapiro-Wilk normality tests were performed. There was equal distribution so a paired correlation test was performed. There was a correlation between the values. In the 1st, 3rd, 5th, 7th variable, the 30, 40, 50 and 60-meter distances, a paired t-test was used to test whether there was a difference between the maximum kilometer speed before and after the strain. In the 2nd, 4th, 6th, 8th variable, the 30, 40, 50 and 60-meter distances, a paired t-test was used to test whether there was a difference between the time before and after the strain. In the 9th variable, the 1RM maximum force test, a paired t-test was used to ascertain whether there was a difference between the maximum left hamstring strength before and after the strain. In the 10th variable, the 1RM maximum force test, a paired t-test was used to ascertain REINTEGRATION PROGRAM FOR PROFESSIONAL FOOTBALL PLAYERS \| G. BOUROGIANNIS ET AL. Reintegration Program (Ferreira et al., 2018) Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 0-10% Match Average N/A 30% Match Average N/A N/A 60% Match Average N/A N/A 60% Match Average N/A N/A 0-63m without sprint distance N/A 190m without sprint distance N/A N/A 380m touching sprint distance N/A N/A 630m achieving maximum speed N/A N/A VHI Exploratory Technical Drills / W Length below 30m to avoid covering considerable amount of VHI distances but allowing the player to achieve it if comfortable. OFF VHI Drills 2x (4x30m) in 5sec. + 1x40m in 6 sec. 30sec active / passive rest between repetitions and 3 min between sets. OFF OFF VHI Drills 2x(4x40m) in 6 sec. + 2x50m in 6sec + 2x40m in 5sec– achieve sprint speed. 30sec active/ passive rest between repetitions and 3 min between sets. OFF OFF VHI Drills w/ maximal intent. 2x(3x50m) in 8sec + 1x50m in 7sec + 1x(4x60m) in 10sec + 1x(3x60m) in 8sec. 30-40sec active/ passive rest between repetitions and 3 min between sets. OFF OFF Start Training with the team Core Stability Recovery Strategies Unloading Legs Legs Maximum Strength Training 85%. Upper- Body Core Stability Recovery Strategies Unloading Legs Upper- Body Core Stability Legs Maximum Strength Training 85%. Upper- Body Core Stability Table 2. Reintegration Program (Ferreira et al., 2018) DOI 10.26773/mjssm.230305 37 37 Results 30 metersh The results showed a significant statistical difference between the maximum mileage for the 30-meter distance before and after the strain, t (7) = 5.694, p = .001. Pre-injury performance of the football players ranged from 19.00 to 22.00 km/h maximum speed. Post-injury with proper rehabilitation the players achieved higher values ranging from 20.00 to 23.00 km/h maximum speed. All the players had higher maximum speed (km/h) in their post-injury test of 30 meters. Figure 1 shows the difference between the maximum speed (km/h) for the 30 m distance before and after the injury. Figure 1. 30-meter distance test before and after injury. Figure 1. 30-meter distance test before and after injury. The results showed a significant statistical difference between the times for the 30-meter distance before and after the strain, t (7) = 5.916, p = .001. Pre-injury performances of the football players ranged from 5 to 5.60 seconds. Post-injury we found that players achieved lower times ranging from 4.70 to 5.30 seconds. All the players had better times in the 30- meter run. between the maximum mileage for the 40-meter distance before and after the fracture, t (7) = 6.328, p = .001. Pre-injury performance of the football players ranged from 20.00 to 22.00 km/h maximum speed . Post-injury with proper rehabilitation players achieved higher values ranging from 21.00 to 27.00 km/h maximum speed. All the football players had higher maximum speed (km/h) in their post-injury test of 40 meters. Figure 2 shows the difference between the maximum speed (km/h) for the 40-meter distance before and after the injury. 40 metersh 40 meters The results showed a significant statistical difference Figure 2. 40-meter distance test before and after injury. Figure 2. 40-meter distance test before and after injury. DOI 10.26773/mjssm.230305 38 REINTEGRATION PROGRAM FOR PROFESSIONAL FOOTBALL PLAYERS \| G. BOUROGIANNIS ET AL. REINTEGRATION PROGRAM FOR PROFESSIONAL FOOTBALL PLAYERS \| G. BOUROGIANNIS ET AL. between the maximum mileage for the 50-meter distance before and after the strain, t (7) = 11.434, p = .001. Pre-injury performance of the football players ranged from 20.50 to 22.50 km/h maximum speed. Post-injury with proper rehabilitation players achieved higher values ranging from 23.00 to 28.00 km/h maximum speed. All the footballers had higher maximum speed (km/h) in their post-injury test of 50 meters. Figure 3 shows the difference between the maximum speed (km/h) for the 50-meter distance before and after the injury. The results showed a significant statistical difference between the times for the 40-meter distance before and after the strain, t (7) = 8.147, p = .001. Pre-injury performance of the football players ranged from 6.60 to 7.29 seconds. Post- injury we found that players achieved lower times ranging from 5.50 to 6.75 seconds. All the players had better time in the 40-meter run. 50 metersh The results showed a significant statistical difference Figure 3. 50-meter distance test before and after injury. Figure 3. 50-meter distance test before and after injury. The results showed a significant statistical difference between the times for the 50-meter distance before and after the strain, t (7) = 15.809, p = .001. Pre-injury performances of the football players ranged from 8.10 to 8.80 seconds. Post- injury we found that the players achieved lower times with values ranging from 6.50 to 7.75 seconds. All the players had better time in the 50-meter test. between the maximum mileage for the 60-meter distance before and after the strain, t (7) = 11.259, p = .001. Pre-injury performance of the football players ranged from 20.40 to 21.60 km/h maximum speed. Post-injury with proper rehabilitation players achieved higher values ranging from 23.00 to 28.00 km/h maximum speed. All the players had higher maximum speed (km/h) in their post-injury test of 60 meters. Figure 4 shows the difference between the maximum speed (km/h) for the 60-meter distance before and after the injury.hif 60 metersh The results showed a significant statistical difference The results showed a significant statistical difference Figure 4. 60-meter distance test before and after injury. Figure 4. 60-meter distance test before and after injury. between the times for the 60-meter distance before and after the strain, t(7) = 16.269, p = .001. Pre-injury performance of the football players ranged from 10 to 10.60 seconds. Post- injury we found that football players achieved lower times ranging from 7.80 to 9.20 seconds. All the players had better times in the 60-meter test. times in the 60-meter test. DOI 10.26773/mjssm.230305 Max Test 1RMh The results showed a significant statistical difference between maximal left hamstring strength before and after DOI 10.26773/mjssm.230305 39 REINTEGRATION PROGRAM FOR PROFESSIONAL FOOTBALL PLAYERS \| G. BOUROGIANNIS ET AL. the strain, t (7) = 4.369, p = .001. Pre-injury performance of the football players ranged from 20 to 40 kg. After injury and appropriate rehabilitation the players achieved higher values ranging from 25 to 45 kg. All of the players had greater peak strength on their tests (1RM) post-injury, so they had better results after injury. According to this study, the football players returned to the exact same pre-injury performance after the rehabilitation – reintegration program. Although the strong point of the present study is the high level of the athletes who were examined and applied the program (Super League 1 professional football players), the results must be interpreted with caution and a number of limitations should be borne in mind. The first limitation is that the sample of the present study was too small. Only eight players were subjected to the tests and followed the reintegration program. Further research is needed with a much larger sample, although this is difficult to achieve when professional players and professional teams are involved. The second limitation of the present study is the distance used to assess and evaluate the players during the reintegration program tests (average high-intensity distance of 630 m at a speed of more than 18 km/h). These distances, as Table 1 shows, were obtained according to data from tests of high-level European professional teams. However, Greece does not belong to the highest class of European professional teams. Obviously, given this limitation, there is a need for research on each individual country group in order to ensure the representativeness of the sample in the European population and make the results of the statistical analysis specific to each country group. Thirdly, it should be noted that the subjective assessment of the participants (subjective assessment of their readiness to return to the team’s competitive activities), using specific self- evaluation questionnaires, was not taken into account. Thus further research is needed with this supplementary element in mind, in order to gather even more information on the players’ readiness to return to the competitive activities of a team and the overall efficiency of a reintegration program. Discussionh The eight professional football players in all variables had better results in both high intensity runs measured using GPS and maximum power. At 30 meters the footballers achieved better times and higher maximum speed (km/h) in their post- injury tests. At 40 meters the footballers also achieved better times and higher maximum speed (km/h) in their post-injury tests. In the 50-meter tests, the footballers also achieved better results in time and maximum speed after the injury than before. The same was true for the 60-meter speed tests. In the right hamstring maximum strength test all footballers had a higher maximum strength in their tests (1RM) after injury. In the left hamstring maximum strength test (1RM) the players also achieved better values after injury than before injury. So the footballers showed an improvement after the injury compared to before. We believe that this improvement occurred due to the right, appropriate and individualized program tailored to each player’s needs that they followed according to the position they played during rehabilitation. With proper rehabilitation and reintegration they recovered to a better level than they were before, improved their weaknesses and finished the season without a relapse or new injury. We believe that prior to the injury the players had deficits in the specific muscle group of the hamstrings. This was the reason they were injured and missed part of the season with a grade 2 strain. Some studies, such as (Woods et al., 2020), disagree with our results: the return of football players to competitive activity after a hamstring strain was not associated with similar high- speed running performance. The deficits observed in high- speed running may be present for many players following a hamstring strain (Orchard et al., 2005). Brooks et al. (2006) found recurrent injuries in the same places, with recurrences at a rate of 23%. This means that something in the whole rehabilitation – reintegration process did not go as it should, leading to frequent recurrences of the hamstring injuries. According to another study (Mendiguchia 1 et al., 2014), the performance of players with a history of hamstring strain was worse after injury than before injury, with a frequent rate of recurrence. Moreover 67% of hamstring injuries in professional football show a post-injury deficit of more than 10%. So it seems that rehabilitation programs are not working properly because if they were there would be no deficit or atrophy. Discussionh All the ratios would be at the percentages they should be (Tol et al., 2014). Although there is a great deal of research on the reintegration of footballers into their previous sporting activity, very few studies concern high-level athletes or professional footballers playing in a country’s top league (Brughelli et al., 2010). Max Test 1RMh Indeed, many professional football teams consider eccentric exercises for the hamstrings the most important for preventing injuries to these muscles (Thorborg et al., 2012). In the injured players, atrophy was observed in the injured limb, once again highlighting the need to strengthen the whole body during the rehabilitation stage and also after the injury, when the players return to training with the rest of the team (Ekstrand J et al., 2015). It is also recommended to increase the number of sets on the injured limb until it is in full balance with the other limb. This is the only way to avoid recurrences or new injuries in the future (Bayer, et al., 2018). DOI 10.26773/mjssm.230305 References Mendez-Villanueva, A., Nuñez, F. J., Lazaro-Ramirez, J. L., Rodriguez-Sanchez, P., Guitart, M., Rodas, G. Martin- Garetxana, I., Lekue, J., Di Salvo, V., & Suarez-Arrones, L. (2022). Knee flexor eccentric strength, hamstring muscle volume and sprinting in elite professional soccer players with a prior strained hamstring. Biology, 11(1), 69. https:// doi.org/10.3390/biology11010069. Altmann, S., Ringhof, S., Neumann, R., Woll, A., & Rumpf, M. C. (2019). Validity and reliability of speed tests used in soccer: A systematic review. PloS one, 14(8), e0220982.h Askling, C. M., Tengvar, M., Saartok, T., & Thorstensson, A. (2007). Acute first-time hamstring strains during high-speed running: a longitudinal study including clinical and magnetic resonance imaging findings. Clin. J. Sports Med., 35(2), 197-206. https://doi. org/10.1177/0363546506294679. https://doi.org/10.1519/JSC.0b013e3181b603ef. Mendiguchia J, & Brughelli M. (2011). A return-to-sport algorithm for acute hamstring injuries. Phys. Ther. Sport, 12(1), 2–14. https://doi.org/10.1016/j.ptsp.2010.07.003. Bayer, M.L., Hoegberget-Kalisz, M., Jensen, M.H., Olesen, J.L., Svensson, R.B., Couppé, C., & Kjaer, M. (2018). Role of tissue perfusion, muscle strength recovery, and pain in rehabilitation after acute muscle strain injury: A randomized controlled trial comparing early and delayed rehabilitation. Scand. J. Med. Sci. Sports, 28(12), 2579– 2591. https://doi.org/10.1111/sms.13269. Mendiguchia, J., Samozino, P., Martinez-Ruiz, E., Brughelli, M., Schmikli, S., Morin, J. B., & Mendez-Villanueva, A. (2014). Progression of mechanical properties during on-field sprint running after returning to sports from a hamstring muscle injury in soccer players. International journal of sports medicine, 35(08), 690-695. https://doi. org/10.1055/s-0033-1363192. Brooks J.H., Fuller C.W., Kemp S.P., & Reddin D.B. (2006). Incidence, risk, and prevention of hamstring muscle injuries in professional rugby union. Am. J. Sports Med., 34(8),1297- 1306. https://doi.org/10.1177/0363546505286022. Mohr M, Krustrup P, & Bangsbo J. (2003). Match performance of high-standard soccer players with special reference to development of fatigue. J. Sports Sci.,21(7), 519-528. https://doi.org/10.1080/0264041031000071182. Brughelli M., Cronin J., Mendiguchia J., Kinsella D., & Nosaka K. (2010). Contralateral leg deficits in kinetic and kinematic variables during running in Australian rules football players with previous hamstring injuries. J. Strength Cond. Res., 24(9), 2539–2544. Orchard J, Best T.M., & Verrall G.M. (2005). Return to play following muscle strains. Clin. J. Sport Med., 15(6), 436- 441. https://doi.org/10.1097/01.jsm.0000188206.54984.65. Schache AG, Dorn TW, Blanch PD, Brown NA, Pandy MG. Mechanics of the human hamstring muscles during sprinting. Med Sci Sports Exerc 2012;44(04):647–658 Carling C, Bradley P, McCall A, Dupont G. Match-to-match variability in high-speed running activity in a professional soccer team. J Sports Sci. 2016 Dec;34(24):2215– 23. Conclusions According to our study, the eight professional football players in all variables had better results in both maximum strength tests and the different speeds they were subjected to. More specifically, in the 30,40,50,60 meters speed tests that we investigated in our team, the players improved their physical performance than before their injury according to the results. Importantly they didn’t present any new injuries. We believe that this is because the rehabilitation program helped them to improve their physical condition and reach a higher level than before their injury, training globally (for all major muscle groups) and more effectively. All the players came back in the second half of the season with the right rehabilitation – In contrast, there were also studies that agreed with ours. They showed that the rehabilitation program they followed not only prevented injuries but also improved the players’ performance at high speeds (Jiménez-Rubio et al., 2019). Another study (Skling et al., 2013) showed potential improvements compared to pre-injury performance in maximum speed, sprinting and high-intensity running. The footballers returned healthy 6 weeks after injury. Another study, however (Mendiguchia J et al., 2011), reports that the players returned neither better nor worse after the injury. 40 DOI 10.26773/mjssm.230305 REINTEGRATION PROGRAM FOR PROFESSIONAL FOOTBALL PLAYERS \| G. BOUROGIANNIS ET AL. injuries: current trends in treatment and prevention. Sports Med., 23(6), 397-404. https://doi.org/10.2165/00007256- 199723060-00005. reintegration at a better level than they had been previously, and finished the season healthy. There was no recurrence or new injury in these players for at least six months. They also played many minutes in official team games, demonstrating the effectiveness of the rehabilitation – reintegration program we implemented. Of course this particular issue needs further research, 40,50,60 meters speed tests could be researched more by other scientists, to see more results and whether they agree or disagree with our research. 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https://openalex.org/W4386600617	https://www.ijfmr.com/papers/2023/5/6159.pdf	English	null	Consumer Buying Behaviour Towards Electric Vehicles in Bengaluru City, Karnataka, India	International Journal For Multidisciplinary Research	2,023	cc-by-sa	4,411	Mr. Tejesh. D1, Dr. Susheela Devi B Devaru2 1Student, Department of MBA, Dr. Ambedkar Institute of Technology, Bengaluru – 560056, Karnataka, India. 2Associate professor, Department of MBA, Dr. Ambedkar Institute of Technology, Bengaluru – 560056, Karnataka, India. ABSTRACT: Electric vehicles are environment friendly powered by electricity, reducing emissions & dependence on fossil fuels. They offer efficient, quiet & smooth driving, contributing to a sustainable transportation. Every day, we come across a wide range of issues and publications that highlight the crucial part that electric vehicles play in changing the face of transportation. In an effort to reduce our dependency on fossil fuels, reduce greenhouse gas emissions, and improve air quality, governments all over the world are moving quickly to implement legislation to stimulate the use of electric vehicles. The dependability & safety of electric vehicles are questioned. In order to analyse how people, perceive the advantages and disadvantages of electric vehicle and the technology they include, this work has been carried out. This paper focuses on consumer buying behaviour towards electric vehicle and the focus of the study is to know the various factors influencing consumer while purchasing elective vehicles. Keywords: Electric vehicle, Consumer buying behaviour, Consumer Attitude, Consumer Perception, Consumer Satisfaction. RESEARCH METHODOLOGY: Descriptive research is a type of research design is used to measure consumer experience, which leads to customer satisfaction. A systematic questionnaire with close - end questions & 5-point Likert scale was developed to examine consumer behaviour. International Journal for Multidisciplinary Research E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com LITERATURE REVIEW: 1. Prof. Tushar Pradhan and Ajaysinh Parmar (2021), A survey on Vadodara city residents' attitudes future electric vehicles the consumers' lack of interest on electric automobiles was noted by the researchers. 2. Vibhuti Pareek, 2022, Perception towards electric vehicles in Indian market: The researcher noted that EV manufacturers need to work on their R & D to improve price range, cost of product, design, style, d b di t f t f bl ti b t EV i I di k t 1. Prof. Tushar Pradhan and Ajaysinh Parmar (2021), A survey on Vadodara city residents' attitudes future electric vehicles the consumers' lack of interest on electric automobiles was noted by the researchers. 1. Prof. Tushar Pradhan and Ajaysinh Parmar (2021), A survey on Vadodara city residents' attitudes future electric vehicles the consumers' lack of interest on electric automobiles was noted by the researchers. 2. Vibhuti Pareek, 2022, Perception towards electric vehicles in Indian market: The researcher noted that EV manufacturers need to work on their R & D to improve price range, cost of product, design, style, and branding to foster a favourable perception about EVs in Indian market. 3. Sajan Acharya (2019), Consumer Perceptions of the Electric Car Industry: Exploring its Contribution to Environmental Sustainability, Researchers noted that focus should be eliminating greenhouse gases and replacing old conventional cars with new electric vehicles to reduce pollutant emissions to keep low. 3. Sajan Acharya (2019), Consumer Perceptions of the Electric Car Industry: Exploring its Contribution to Environmental Sustainability, Researchers noted that focus should be eliminating greenhouse gases and replacing old conventional cars with new electric vehicles to reduce pollutant emissions to keep low. OBJECTIVE OF THE STUDY: 1) To study consumers buying behaviour towards electric vehicles in Bengaluru city. 2) To study factors influencing consumer while purchasing elective vehicles. 3) To determine the affordability of pricing that consumer may pay for an electric/hybrid vehicle. 4) To study government initiatives taken to promote electric/hybrid vehicles & subsidies provided on electricity. 4) To study government initiatives taken to promote electric/hybrid vehicles & subsidies provided on electricity. STATEMENT OF THE PROBLE: People are beginning to think about protecting the environment as much as they can because environmental problems are prevalent in India. Fuels like gasoline and diesel are well known. People are switching to electric vehicles as a result of the majority of environmental harm coming from transportation. Despite the fact that the EV idea is currently fully established, few individuals are aware of or confident in it. The dependability and safety of electric cars are questioned by them. In order to analyse how people, perceive the advantages and disadvantages of electric vehicle and the technology they include, this work has been carried out. INTRODUCTION: Electric vehicles (EVs) have gained popularity as eco-friendly alternatives to gas-powered cars. They run on electricity, can be charged externally, and offer benefits like reduced reliance on fossil fuels and lower maintenance costs. This makes them a solution to environmental issues like pollution and climate change. The surge in EVs is driven by concerns about urban air pollution, with many major Indian cities among the world's most polluted areas. Transportation emissions are a significant contributor, prompting manufacturers and governments to explore eco-friendly options. Governments worldwide are endorsing electric mobility as a way to combat pollution. For example, China promotes hybrid vehicles, while the UK incentivizes EV purchases in cities like London. In India, electric three-wheelers are popular, but the power distribution network needs improvement. However, there's potential for EV growth, especially in electric bikes and four-wheelers, if the government invests in charging infrastructure and incentives. India aims for an all-electric vehicle fleet by 2030, with the Minister of Road Transport and Highways working with the Society of Indian Automobile Manufacturers to promote this transition. This reflects the government's commitment to sustainable transportation. Volume 5, Issue 5, September-October 2023 IJFMR23056159 1 HYPOTHESIS OF THE STUDY H0 : There is no significant, relationship b/w mileage of electric vehicle & electric vehicle company preferred. H1 Th i i ifi t l ti hi b/ il f EV & l t i hi l f d H0 : There is no significant, relationship b/w mileage of electric vehicle & electric vehicle company preferred. H1 : There is significant relationship b/w mileage of EV & electric vehicle company p SAMPLE SIZE The sample size for the study is set at 200 people. Due to time constraints, only 200 people will be surveyed. The sample size for the study is set at 200 people. Due to time constraints, only 200 people will be surveyed. SAMPLING METHOD Random sampling has been used because the selection of units from the population has been done randomly by visiting around 15 - 20 EV outlets in nagarbhavi and convenience sampling was done based on availability and accessibility by forwarding structured questionnaire through google forms at our convenient level. POPULATION & SAMPLING UNIT Research & survey activities had been done for the population of Bangalore city in order to know buying behaviour towards Electric vehicle and services in Bangalore. Sampling Units - Research has been done for both male and female respondents using electric vehicles. STATISTICAL DESIGN The collected data is tabulated and analysed using statistical procedures. such as percentage analysis. To determine the association between two variables, SPSS software is utilized. The data is displayed using percentage and bar chart analysis, which aids in judging the level of customer behavior by accurately showing factors.  Table No. 1 Table showing age of respondents. Age No of respondents Percentage of respondent 18 - 25 50 25 % 25 - 30 66 33 % 30 - 40 66 33 % 40 & Above 18 9 % Total 200 100 % A l i  Table No. 1 SOURCES OF DATA COLLECTION  Primary data Primary data are collected with specific set of objectives to assess buying behaviour of the consumers. The first-hand information was collected from respondents in nagarbhavi, Bangalore city by visiting EV outlets & forwarding structured questionnaire through google forms. Primary data are collected with specific set of objectives to assess buying behaviour of the consumers. The first-hand information was collected from respondents in nagarbhavi, Bangalore city by visiting EV outlets & forwarding structured questionnaire through google forms.  Secondary data Secondary data is been collected from different sources including websites, journals, articles, magazines etc… IJFMR23056159 Volume 5, Issue 5, September-October 2023 2 y It can be analysed that out of 200 respondents, 25% of respondents are 18 - 25 years, 33% of responndents are between 25 - 30 years, 33% of respondents are between 30 - 40 years and 9 % of respondents are 40 years and above.  Table No. 2 Analysis: ational Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com  Graph No. 1 Graph showing age of the respondents. Inference: From the above graph, it can be construed that respondents who belongs to 25-30 age group & 30-40 age group are the major customers towards EV, the reason behind this is the young working proffessionals who fall under this age group tends to prefer Luxury lifesytle. Hence consumers of this age group prefers electric vehicles. 50 66 66 18 200 25% 33% 33% 9% 100% 18 - 25 25 - 30 30 - 40 40 & ABOVE TOTAL Age No of respondents  Graph No. 1 Graph showing age of the respondents. Graph showing age of the respondents. Graph showing age of the respondents. Inference: : above graph, it can be construed that respondents who belongs to 25-30 age group From the above graph, it can be construed that respondents who belongs to 25-30 age group & 30-40 age group are the major customers towards EV, the reason behind this is the young working proffessionals who fall under this age group tends to prefer Luxury lifesytle. Hence consumers of this age group prefers electric vehicles. From the above graph, it can be construed that respondents who belongs to 25-30 age group & 30-40 age group are the major customers towards EV, the reason behind this is the young working proffessionals who fall under this age group tends to prefer Luxury lifesytle. Hence consumers of this age group prefers electric vehicles.  Table No. 2 Table showing Profession of the respondent. Profession No of respondents Percentage of respondent Student 30 15% Employee 92 46% Professional 23 11.5% Self - Employed 47 23.5% Housewife 7 3.5% Retired 1 0.5% Total 200 100 % Analysis: It can be analysed that out of 200 respondents, majority 46 % of respondents are Employed, 23.5 % of respondents are Self – Employed. Hence, they are preferring the Electric vehicles. Analysis: It can be analysed that out of 200 respondents, 25% of respondents are 18 - 25 years, 33% of responndents are between 25 - 30 years, 33% of respondents are between 30 - 40 years and 9 % of respondents are 40 years and above. It can be analysed that out of 200 respondents, 25% of respondents are 18 - 25 years, 33% of responndents are between 25 - 30 years, 33% of respondents are between 30 - 40 years and 9 % of respondents are 40 years and above. Volume 5, Issue 5, September-October 2023 IJFMR23056159 3 Analysis: It can be analysed that majority 42% respondents are preferring travelling range of 200 – 350 km, 23% respondents are preferring travelling range of 350 – 500 km etc.  Table No. 3  Table No. 3 Table showing mileage preferred by respondents in 0ne complete charge. Mileage preferred in 0ne complete charge No of respondents Percentage of respondent 0 – 100 km 30 15% 100 – 200 km 26 13% 200 – 350 km 84 42% 350 – 500 km 46 23% 500 km & above 14 7% Total 200 100% Table showing mileage preferred by respondents in 0ne complete charge. rnational Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com  Graph No. 2 Graph showing Profession of the respondent. Graph showing Profession of the respondent. Inference: F th b h it b t d th t l th j t th b hi d thi 30 92 23 47 7 1 200 STUDENT EMPLOYEE PROFESSIONAL SELF - EMPLOYED HOUSEWIFE RETIRED TOTAL Profession Inference: 30 92 23 47 7 1 200 STUDENT EMPLOYEE PROFESSIONAL SELF - EMPLOYED HOUSEWIFE RETIRED TOTAL Profession Inference: From the above graph, it can be construed that employees are the major customers, the reason behind this is, nowadays people are more towards Travelling & new technologies. Though EV outlets are located everywhere in Bangalore which make consumer more convenient to visit the outlet. So, they do visit more to the EV outlets. From the above graph, it can be construed that employees are the major customers, the reason behind this is, nowadays people are more towards Travelling & new technologies. Though EV outlets are located everywhere in Bangalore which make consumer more convenient to visit the outlet. So, they do visit more to the EV outlets. Analysis: y It can be analysed that out of 200 respondents, majority 46 % of respondents are Employed, 23.5 % of respondents are Self – Employed. Hence, they are preferring the Electric vehicles. It can be analysed that out of 200 respondents, majority 46 % of respondents are Employed, 23.5 % of respondents are Self – Employed. Hence, they are preferring the Electric vehicles. Volume 5, Issue 5, September-October 2023 IJFMR23056159 4 International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com  Graph No. 2 Graph showing Profession of the respondent. Inference: From the above graph, it can be construed that employees are the major customers, the reason behind this is, nowadays people are more towards Travelling & new technologies. Though EV outlets are located everywhere in Bangalore which make consumer more convenient to visit the outlet. So, they do visit more to the EV outlets. 30 92 23 47 7 1 200 STUDENT EMPLOYEE PROFESSIONAL SELF - EMPLOYED HOUSEWIFE RETIRED TOTAL Profession  Graph No. 3  Graph No. 3 Graph showing respondents travelling km’s perfered in 0ne complete charge. Graph No. 3 Graph showing respondents travelling km’s perfered in 0ne complete charge. p Graph showing respondents travelling km’s perfered in 0ne complete charge. Inference: From the above graph, it can be construed that majority of respondents prefer vehicles with a range between 200 and 350 km on one complete charge. However, there is also a significant interest in vehicles with ranges of 0 – 100 km and 350 – 500 km. 30 26 84 46 14 200 0 – 100 KM 100 – 200 KM 200 – 350 KM 350 – 500 KM 500 KM & ABOVE TOTAL Mileage perfered in one complete charge p g p g p p g Inference: 30 26 84 46 14 200 0 – 100 KM 100 – 200 KM 200 – 350 KM 350 – 500 KM 500 KM & ABOVE TOTAL Mileage perfered in one complete charge Inference: 30 26 84 46 14 200 0 – 100 KM 100 – 200 KM 200 – 350 KM 350 – 500 KM 500 KM & ABOVE TOTAL Mileage perfered in one complete charge Inference: From the above graph, it can be construed that majority of respondents prefer vehicles with a range between 200 and 350 km on one complete charge. However, there is also a significant interest in vehicles with ranges of 0 – 100 km and 350 – 500 km. From the above graph, it can be construed that majority of respondents prefer vehicles with a range between 200 and 350 km on one complete charge. However, there is also a significant interest in vehicles with ranges of 0 – 100 km and 350 – 500 km. ble No. 4 Table showing most preferred Electric Vehicles company by the respondents  Table No. 4 Table showing most preferred Electric Vehicles company by the respondents. Most preferred Electric Vehicles company No of respondents Percentage of respondent Tata motors 52 26% Hyundai 24 12% Hero electric 12 6% TVS 17 8.5% Ather electric 26 13% Ola electric 15 7.5% Mahindra electric 23 11.5% Others 31 15.5% Total 200 100% Table showing most preferred Electric Vehicles company by the respondents. Analysis: y It can be analysed that majority 42% respondents are preferring travelling range of 200 – 350 km, 23% respondents are preferring travelling range of 350 – 500 km etc. Volume 5, Issue 5, September-October 2023 IJFMR23056159 5 International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com Graph No. 4 Graph showing most preferred Electric Vehicles company by the respondents. Graph showing most preferred Electric Vehicles company by the respondents 52 24 12 17 26 15 23 31 200 TATA MOTORS HERO ELECTRIC ATHER ELECTRIC MAHINDRA ELECTRIC TOTAL Most preferred Electric Vehicles company Inference: From the above graph, it can be construed that Tata Motors emerged as the most preferred EV company among the respondents, based on the highest number of respondents selecting it as their preferred choice in the survey. followed by Hyundai, indicating their popularity and market appeal in the electric vehicle segment. Analysis: It can be analysed that majority 26% of them prefer tata motors,15.5% of respondents prefer other companies which is not mentioned in the list,13% of respondents choose Ather electric bikes,12% select Hyundai company,11.5% prefers Mahindra electric vehicle,8.5% of consumers prefer TVS,7.5% prefers ola & 6% of them choose Hero electric vehicle. Volume 5, Issue 5, September-October 2023 IJFMR23056159 6 International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com ❖ FINDINGS: 1. It can be analysed that out of 200 respondents, 25% of respondents are 18 - 25 years, 33% of respondents are between 25 - 30 years, 33% of respondents are between 30 - 40 years and 9 % of respondents are 40 years and above. 1. It can be analysed that out of 200 respondents, 25% of respondents are 18 - 25 years, 33% of respondents are between 25 - 30 years, 33% of respondents are between 30 - 40 years and 9 % of respondents are 40 years and above. 2. It can be analysed that out of 200 respondents, majority 46 % of respondents are Employed, 23.5 % of respondents are Self – Employed. Hence, they are preferring the Electric vehicles. 2. It can be analysed that out of 200 respondents, majority 46 % of respondents are Employed, 23.5 % of respondents are Self – Employed. Hence, they are preferring the Electric vehicles. 3. It can be analysed that majority 46% of respondents know about EV from social media, 27% of respondents knew from television, 15.5% of respondents knew from Local observation & 11.5% from friend/relatives. 3. It can be analysed that majority 46% of respondents know about EV from social media, 27% of respondents knew from television, 15.5% of respondents knew from Local observation & 11.5% from friend/relatives. 4. It can be analysed that majority 33.5% of respondents are willing to switch to protect environment, 33% of respondents are switching due to increased price of petrol & diesel vehicle & 20.5% of respondents are all of the above & 13% of respondents are switching because of better driving experience. 5. It can be analysed that majority 39.5% of respondents are willing to spend between 10 – 25 Lakhs,27.5% are spending below 5 Lakhs,25% of respondents are willing to spend between 5 – 10 Lakhs,7% of respondents are willings to spend between 25 – 50 Lakhs, 1% of respondents are spending above 50 Lakhs. 6. It can be analysed that majority 44% of respondents are preferring car,28.5% are preferring Bike,14% of them are preferring scooter & 13.5% of respondents are preferring other alternatives. 7. It can be analysed that majority 42% respondents are preferring travelling range of 200 – 350 km, 23% respondents are preferring travelling range of 350 – 500 km etc. 8. ❖ FINDINGS: It can be analysed that majority 26% of them prefer tata motors,15.5% of respondents prefer other companies which is not mentioned in the list,13% of respondents choose Ather electric bikes,12% select Hyundai company,11.5% prefers Mahindra electric vehicle,8.5% of consumers prefer TVS,7.5% prefers ola & 6% of them choose Hero electric vehicle. HYPOTHESIS TESTING (CORREALATION) IJFMR23056159 Volume 5, Issue 5, September-October 2023 7 HYPOTHESIS TESTING (CORREALATION) H0 : There is no significant, relationship b/w mileage of electric vehicle & electric vehicle company preferred. H1 : There is significant relationship b/w mileage of EV & electric vehicle company preferred INTERPRETATION P value = 0.020 Level of significance = 0.05 (P value 0.020 < LOS = 0.05) ✓ Pearson product correlation relationship was found moderately positive and statistically significant. ✓ Therefore, mileage is having significant relationship with Electric’ Vehicle company preferred. ✓ Hence, there is significant relationship b/w mileage of electric vehicle & electric vehicle company preferred. How much Mileage do you prefer in one complete charge? Which Electric Vehicles company would you prefer the most? How much mileage do you perfer in one complete chagre? Pearson Correlation 1 .020 Sig. (2-tailed) .782 N 200 200 Which Electric Vehicles company would you prefer the most? Pearson Correlation .020 1 Sig. (2-tailed) .782 N 200 200 ( ) H0 : There is no significant, relationship b/w mileage of electric vehicle & electric vehicle company preferred. eferred. 1 : There is significant relationship b/w mileage of EV & electric vehicle company preferred NTERPRETATION How much Mileage do you prefer in one complete charge? Which Electric Vehicles company would you prefer the most? How much mileage do you perfer in one complete chagre? Pearson Correlation 1 .020 Sig. (2-tailed) .782 N 200 200 Which Electric Vehicles company would you prefer the most? Pearson Correlation .020 1 Sig. (2-tailed) .782 N 200 200 INTERPRETATION P value = 0.020 Level of significance = 0.05 (P value 0.020 < LOS = 0.05) ✓ Pearson product correlation relationship was found moderately positive and statistically significant. ✓ Therefore, mileage is having significant relationship with Electric’ Vehicle company preferred. ✓ Hence, there is significant relationship b/w mileage of electric vehicle & electric vehicle company preferred. Volume 5, Issue 5, September-October 2023 IJFMR23056159 7 International Journal for Multidisciplinary Research (IJFMR) E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com ❖ SUGGESTIONS The number of charging stations need to be expanded, as more people are interested in buying EVs. Electric vehicles should be prioritized in order to reduce pollution and greenhouse gas emissions. Companies should concentrate on informing the public about new electric vehicle modes. Petrol prices are steadily rising. Electric vehicles have the potential to help solve the problem of rising gasoline prices. Electric car promotion by the government will benefit the country's future growth. To encourage the adoption of electric vehicles, the government should provide incentives and subsidies. Reduced tax rates can tempt some buyers to choose electric vehicles. By lowering the initial cost of electric vehicles, the industry will grow in near future. However, to accelerate the EV revolution, a multi-faceted approach is essential. This includes investing in a robust charging infrastructure network that caters to both urban and rural areas, fostering collaborations between public and private sectors, and raising awareness among consumers about the economic and environmental advantages of EVs. Moreover, supporting research and development for advanced battery technologies and localized production will be crucial in making EVs more affordable and accessible to a wider segment of the population. ❖ CONCLUSION: The analysis, of the survey data’ provides valuable insights into attitudes and preferences of potential electric vehicle (EV) consumers. The study reveals a gender disparity in EV interest, emphasizing need for targeted marketing strategies, to engage more female consumers. Age groups, particularly 25-30 and 30-40, exhibit a strong inclination towards EVs, driven by young professionals seeking a luxury lifestyle. Inc0me level does not significantly’ influence EV preference, with the 25k-40k income range showing notable interest. Graduation and post-graduation holders are well-informed about branded EV outlets and exhibit brand differentiation. Environmental concerns, government initiatives, and rising fuel prices play vital roles in motivating EV adoption. Addressing barriers like range anxiety, charging time, and limited choice is pivotal. Cars are the favored EV type, Tata Motors and Hyundai lead in preference, emphasizing the need for enhanced brand appeal. Respondents prioritize EVs with ranges of 200-350 km, indicating a positive market outlook. Government policies and incentives receive favorable feedback, highlighting effective support. Incentives, perceived savings, and environmental benefits influence purchasing decisions. To promote EV adoption, targeting different demographics, addressing concerns, and creating affordable, high-quality EVs are key. Public education can dispel misconceptions. In sum, this analysis underscores the importance of targeted marketing, addressing barriers, and continued government support for a thriving EV market. Volume 5, Issue 5, September-October 2023 IJFMR23056159 8 8 ❖ REFERENCES: 1. Janardan Prasad Kesari,Y.S.(2019). Opportunities and Scope for Electric Vehicles in India. IJME Journal,8. 1. Janardan Prasad Kesari,Y.S.(2019). Opportunities and Scope for Electric Vehicles in India. IJME Journal,8. 2. Nimesh, V., Sharma, D., Reddy, V. M., & Goswami, A. K. (2020). Implication viability assessment of shift to electric vehicles for present power generation scenario of India. Energy, 195, 116976. https://doi.org/10.1016/j.energy.2020.116976 3. Marketing Management 14e, A South Asian Perspective, Philip Kotler, Kevin Keller, Abraham Koshy, Mithileshwar Jha, Pearson. 3. Marketing Management 14e, A South Asian Perspective, Philip Kotler, Kevin Keller, Abraham Koshy, Mithileshwar Jha, Pearson. 4. Electric Vehicle Sales India – FY2020 \| Team Auto Punditz \| Apr 20, 2020 \| AUTO PUNDITZ \| https://www.autopunditz.com/post/electric-vehicle-sales-india-fy2020 4. Electric Vehicle Sales India – FY2020 \| Team Auto Punditz \| Apr 20, 2020 \| AUTO PUNDITZ \| https://www.autopunditz.com/post/electric-vehicle-sales-india-fy2020 5. Egbue, O., & Long, S. (2012). Barriers to widespread adoption of electric vehicles: An analysis of consumer attitudes and perceptions. Energy policy, 48, 717-729. 5. Egbue, O., & Long, S. (2012). Barriers to widespread adoption of electric vehicles: An analysis of consumer attitudes and perceptions. Energy policy, 48, 717-729. 6. Kotler, P. (2011). Marketing Management, 11th edition, Prentice-Hall India. 7. Michael R. Solomon (2003). Consumer Behavior, 6th edition, Upper Saddle River, N.J: Pearson Prentice Hall, pp. 199-202. 7. Michael R. Solomon (2003). Consumer Behavior, 6th edition, Upper Saddle River, N.J: Pearson Prentice Hall, pp. 199-202. 8. Consumer Behavior Towards Buying Electric Vehicles \| PDF \| Electric Vehicle \| Car (scribd.com) 8. Consumer Behavior Towards Buying Electric Vehicles \| PDF \| Electric Vehicle \| Car (scribd.com) 9. NITI Aayog & World Energy Council.Zero Emission Vehicles (ZEVs):Towards a Policy Framework, 2018 9. NITI Aayog & World Energy Council.Zero Emission Vehicles (ZEVs):Towards a Policy Framework, 2018 10. Marketing Management, Arun Kumar &Meenakshi N, 2/e, Vikas, 2012 10. Marketing Management, Arun Kumar &Meenakshi N, 2/e, Vikas, 2012 10. Marketing Management, Arun Kumar &Meenakshi N, 2/e, Vikas, 2012 11. Consumer Behavior- Satish K. Batra& S H HKazmi, Excel Books. 11. Consumer Behavior- Satish K. Batra& S H HKazmi, Excel Books. IJFMR23056159 Volume 5, Issue 5, September-October 2023 9
https://openalex.org/W2789131406	https://europepmc.org/articles/pmc5799194?pdf=render	English	null	Impaired Wnt signaling in dopamine containing neurons is associated with pathogenesis in a rotenone triggered Drosophila Parkinson’s disease model	Scientific reports	2,018	cc-by	9,783	Impaired Wnt signaling in dopamine containing neurons is associated with pathogenesis in a rotenone triggered Drosophila Parkinson’s disease model Received: 25 August 2017 Accepted: 25 January 2018 Published: xx xx xxxx Received: 25 August 2017 Accepted: 25 January 2018 Published: xx xx xxxx Flora Stephano1,3, Stella Nolte1,6, Julia Hoffmann1,7, Samar El-Kholy1,4, Jakob von Frieling1 Iris Bruchhaus2, Christine Fink1,5 & Thomas Roeder1,5 Parkinson’s disease, which is the one of the most common neurodegenerative movement disorder, is characterized by a progressive loss of dopamine containing neurons. The mechanisms underlying disease initiation and development are not well understood and causative therapies are currently not available. To elucidate the molecular processes during early stages of Parkinson’s disease, we utilized a Drosophila model. To induce Parkinson’s disease-like phenotypes, we treated flies with the pesticide rotenone and isolated dopamine producing neurons of animals that were at an early disease stage. Transcriptomic analyses revealed that gene ontologies associated with regulation of cell death and neuronal functions were significantly enriched. Moreover, the activities of the MAPK/EGFR- and TGF-β signaling pathways were enhanced, while the Wnt pathway was dampened. In order to evaluate the role of Wnt signaling for survival of dopaminergic neurons in the disease model, we rescued the reduced Wnt signaling activity by ectopic overexpression of armadillo/β-catenin. This intervention rescued the rotenone induced movement impairments in the Drosophila model. Taken together, this initial study showed a highly relevant role of Wnt signaling for dopamine producing neurons during pathogenesis in Parkinson’s disease and it implies that interfering with this pathway might by a suitable therapeutic option for the future. Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder with an incidence of about 1% in people older than 65 years1,2. Patients suffering from PD show a selective and progressive loss of dopaminergic (DA) neurons and the presence of Lewy bodies in surviving neurons in the Substantia nigra3. The most important symptoms are motor system disturbances such as bradykinesia, resting tremor, rigidity and postural instability. These symptoms appear when degeneration of DA neurons is at an advanced stage. Currently, no causative treat- ment strategy is available for this complex disease, where both, genetic susceptibility and environmental factors contribute to induce pathogenesis. Although epidemiological and genetic studies yielded important insights into causes underlying pathogenesis, the precise pathologic molecular mechanisms remain unclear1,4–6.hi y g p g p p g The identification of genes causatively related to familiar forms of the disease raised the hope to identify common cellular signatures that underlie pathogenesis7,8. Received: 25 August 2017 Accepted: 25 January 2018 Published: xx xx xxxx www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Impaired Wnt signaling in dopamine containing neurons is associated with pathogenesis in a rotenone triggered Drosophila Parkinson’s disease model However, a detailed understanding of the first steps that trigger Parkinson’s disease (PD) is currently almost completely missing. The major strategy followed by most researchers in the field aims to understand the effects caused by mutations in genes linked to PD including Parkin, Pink, SCNA or LRRK2. Alternatively, a detailed understanding of common signaling pathways that are 1Kiel University, Zoological Institute, Department Molecular Physiology, Kiel, Germany. 2Bernhard-Nocht Institute for Tropical Medicine, Hamburg, Germany. 3University of Dar es Salaam, Department of Zoology and Wildlife Conservation, Dar es Salaam, Tanzania. 4Tanta University, Faculty of Science, Department of Zoology, Tanta, Egypt. 5Airway Research Center North of the German Center for Lung Research (DZL), Grosshansdorf, Germany. 6Present address: Aarhus University, Dandrite, Department Molecular Biology and Genetics, Aarhus, Denmark. 7Present address: Amedon AG, Lübeck, Germany. Correspondence and requests for materials should be addressed to T.R. (email: troeder@zoologie.uni-kiel.de) SCIENTIFIC REPOrTS \| (2018) 8:2372 \| DOI:10.1038/s41598-018-20836-w 1 www.nature.com/scientificreports/ deregulated during pathogenesis might pave the way for a better understanding of disease development. A very promising candidate that appears to be deregulated during disease development is the canonical Wnt pathway. Evidences that link deregulated Wnt signaling with a number of neurological disorders have already been high- lighted9,10. In Alzheimer’s disease (AD), an impairment of canonical Wnt signaling has been tightly associated with early disease phases. There, amongst other pathway components, Dickkopf-1 (Dkk1) levels are enhanced, which effectively inhibits Wnt signaling11. Other risk factors for AD such as specific ApoE4- or LRP6 variants are also known to reduce neuronal Wnt signaling12. Although, an association between early stages of the disease and impaired Wnt signaling has also been shown in PD8,13,14, it is not generally accepted that deregulated Wnt signaling is indeed a driving force underlying PD development. To study these early molecular events in DA neurons that finally lead to PD, animal models have been the foremost tools. Rodent models of PD, especially murine ones have been extremely successful, despite the fact that they do not recapitulate all facets of the dis- ease15. Thus, alternative animal models, including those utilizing invertebrates are highly relevant. Very simple and genetically tractable model became very attractive with a series of different Drosophila model being the most successful ones16. Targeted overexpression of human alpha-synuclein induces symptoms typically seen in human PD patients similarly as other PD susceptibility genes did17–19. Results R Rotenone induced changes in locomotor behavior in an administration time dependent man- ner. This study aims to elucidate the molecular changes occurring in DA neurons during early phases of dis- ease development. PD like symptoms were induced by rotenone application. Previous studies have shown that detectable motor impairments and DA neuron loss start after chronic exposure with rotenone for several days and that this symptomatic phase starts after the majority of DA neurons were already severely damaged20,23. This severe motor impairment is known to be not associated with a concurrent demise of dopaminergic cells24. In order to monitor the onset and development of the PD-associated pathologies, we used our experimental animals (F1 of TH-GAL4 X 20xUAS-mCD8::GFP) and treated them with rotenone (Fig. 1). Brains of non-treated animals (Fig. 1A,A′) and of rotenone treated (0.5 mM for 10 d; Fig. 1B,B′) were analyzed. Shown are two regions per treatment type (control, A,A′; rotenone for 10d, B,B′) of representative brains of the above mentioned genotype (TH-GAL4 X 20xUAS-mCD8::GFP) labeled with anti-GFP antibodies. The TH-Gal4 driver line labels about 50% of all dopaminergic cells in the fly’s brain25. The number and location of neurons remained mostly unchanged, which was in line with previous observations24. In non-treated control animals, we observed a mean of 52.13 (±2.1, S.E.M) GFP-positive cells per hemisphere, whereas we identified 49.5 (±5.3, S.E.M.) cells per hemisphere in rotenone- treated animals. This difference was not statistically significant (p = 0.92, N = 8). In contrast, treat- ment with the sublethal dose of 0.5 mM rotenone had a significant effect on the motor abilities of the treated flies. We quantified their ability to climb a vertical plane (Fig. 1C). There was no obvious locomotor deficit observed in flies exposed to rotenone for up to 3 days. From day 6 onwards, flies showed statistically reduced locomotor ability in comparison to untreated flies, meaning that their mean climbing distance was reduced substantially (Fig. 1C). Thus, we choose flies treated for three days with rotenone for all subsequent transcriptomic analyses. At this time point, we can assume that the first signs of pathological alterations had been induced, but still before the onset of the phenotypical hallmark, the locomotor impairment, of this disease model. Impaired Wnt signaling in dopamine containing neurons is associated with pathogenesis in a rotenone triggered Drosophila Parkinson’s disease model Moreover, simple, pharmacologically induced PD model that utilize either the pesticide rotenone or paraquat to trigger DA neuronal impairment were introduced in flies. They showed a very impressive phenotype, a substantially reduced and quantifiable climbing ability20. The different Drosophila models of PD have been very informative in order to identify mechanisms underlying disease development and potential interventions that delay or prevent it. Very impressive were the observations that either coffee and cigarette smoke induce Nrf2-mediated protection in these models of PD21,22. f g p In the current study, we focused on understanding molecular responses in DA neurons during the early phase of PD, where impairments of the motor abilities are still not detectable, in order to identify potential targets for therapeutic intervention. We used rotenone to induce Parkinsonism in the fly and combined this with a cDNA microarray analysis of DA neurons that were isolated using a magnetic bead based approach. Our data provide evidences that the outcomes of various highly relevant molecular signaling pathways are modified in DA neu- rons that are in an early stage of PD. Amongst these affected systems are the Wnt-, MAPK/EGFR-, TGF-β-, and TOR-signaling pathways, which are known to be important for cell survival and/or cell death in the CNS. Results R Consequently, we performed whole transcriptome analysis with RNA isolated specifically from DA neurons after this time of treatment to ensure that those effects can be identified that occur prior to development of the disease associated phenotype. In parallel, parts of the experimental populations were used for phenotypical analyses of the rotenone induced Parkinsonism over time, which always showed the kinetics as outlined in Fig. 1C. In order to isolate the mCD8::GFP tagged neurons, they were decollated first (Fig. 1D). The figure shows few GFP-positive cells (TH-Gal4 positive) and numerous other, non-labeled neurons. Using an isolation protocol based and anti-mCD8 coupled magnetic beads, the mCD8-GFP-tagged cells were isolated and all non-labeled cells depleted from the preparation. The high degree of enrichment of mCD8-GFP-positive cells is shown in Fig. 1E, where some GFP-positive cells (green arrows), but no non-labeled cells are detectable. Rotenone induced gene expression changes in DA neurons in an early phase of PD. Although flies treated with 0.5 mM rotenone for three days appear to coordinate their movement normally (Fig. 1B), a number of genes show differential expression in DA neurons obtained from rotenone treated animals compared with those isolated from sham-treated ones. Expression of 765 genes was up-regulated (>1.5 fold) and that of 357 genes was down-regulated (<0.67 fold) if compared with the signals obtained from DA neurons isolated from sham-treated animals. To further filter and identify relevant regulated genes in the two lists, we used different Gene Ontology (GO) analysis programs such as the DAVID26 and the GOrilla program packages27. Different GO SCIENTIFIC REPOrTS \| (2018) 8:2372 \| DOI:10.1038/s41598-018-20836-w 2 www.nature.com/scientificreports/ p / Figure 1. Isolation of dopamine producing cells following rotenone treatment. Brains of adult animals of the genotype TH-GAL4 > 20xUAS-mCD8::GFP from control animals (A,A′) as well as from animals treated for 10 d with rotenone (B,B′) were analyzed using anti GFP immunohistochemistry. GFP-positive, dopamine containing neurons are shown (arrows) in two different Z-areas (A,A′,B,B′). (C) Exposure of rotenone for different periods induced climbing disability in a time dependent manner. The distance reached by animals climbing a vertical plane within 20 s is given. Shown are mean values of three independent experiments, ±SEM, n = 20 individuals for each group (P < 0.01, p < 0.005). (D,E) Magnetic bead assisted isolation dopamine containing cells using beads coated with anti-mCD8 antibodies. Results R Functional categories overrepresented in the lists of genes differentially expressed in rotenone treated flies identified by DAVID (Table 1, p < 0.05). (C) Fold changes for genes associated with the GO term regulation of apoptosis (D) Oxidative phosphorylation (E) neurotransmitter secretion (F) ageing. Error bars represent SEM. Damm: Death associated molecule related to Mch2, Dcp-1: Death caspase 1, BI-1: Bax Inhibitor 1, Jafrac2: Thioredoxin peroxidase 2, th: thread, PDCD-5: Programmed cell death 5, Hsp60D, out: Outsiders, sox15: Sox box protein 15, Prx2540-2: Peroxiredoxin 2540-2, Cyp6a2, Cyp12a5, Cyp12a4: Cytochrome p450-6a2, 12a4, 12a5, respectively, Su(Tpl): CoVIIc: Cytochrome c oxidase subunit VIIc, CG6463, Csp: Cysteine string protein, Fas2: Fasciclin2, Frq1: Frequenin 1, Hsc70-4: Heat shock protein cognate 4, Sap47: Synapse associated protein 47kD, Syx1A: Syntaxin 1A, EndoA: Endophilin A, Usnp: Ubisnap, clumsy, and MstProx, Ef1alpha 48D: Elongation factor 1 alpha 48D, Atpalpha: Sodium pump alpha subunit, Dad: daughter against dpp, alpha-Man-1: alpha- Mannosidase class 1a and Mthl2: methuselah-like 2. Figure 2. Early transcriptional response to rotenone treatment of dopamine containing cells. GO analyses of genes that were upregulated (A) or downregulated (B) in dopamine containing cells in response to rotenone treatment. Clustering was performed with the DAVID program package68. Functional categories overrepresented in the lists of genes differentially expressed in rotenone treated flies identified by DAVID (Table 1, p < 0.05). (C) Fold changes for genes associated with the GO term regulation of apoptosis (D) Oxidative phosphorylation (E) neurotransmitter secretion (F) ageing. Error bars represent SEM. Damm: Death associated molecule related to Mch2, Dcp-1: Death caspase 1, BI-1: Bax Inhibitor 1, Jafrac2: Thioredoxin peroxidase 2, th: thread, PDCD-5: Programmed cell death 5, Hsp60D, out: Outsiders, sox15: Sox box protein 15, Prx2540-2: Peroxiredoxin 2540-2, Cyp6a2, Cyp12a5, Cyp12a4: Cytochrome p450-6a2, 12a4, 12a5, respectively, Su(Tpl): CoVIIc: Cytochrome c oxidase subunit VIIc, CG6463, Csp: Cysteine string protein, Fas2: Fasciclin2, Frq1: Frequenin 1, Hsc70-4: Heat shock protein cognate 4, Sap47: Synapse associated protein 47kD, Syx1A: Syntaxin 1A, EndoA: Endophilin A, Usnp: Ubisnap, clumsy, and MstProx, Ef1alpha 48D: Elongation factor 1 alpha 48D, Atpalpha: Sodium pump alpha subunit, Dad: daughter against dpp, alpha-Man-1: alpha- Mannosidase class 1a and Mthl2: methuselah-like 2. terms were found to be enriched in either the cohorts of up- or down-regulated genes or of both groups together. Results R (D) Cell suspension of decollated neurons containing GFP-positive (green arrows) as well as non-labeled neurons (white arrows). (E) GFP- positive cells (green arrows) and magnetic beads (white arrow heads) after bead-assisted isolation devoid of non-labeled cells. Figure 1. Isolation of dopamine producing cells following rotenone treatment. Brains of adult animals of the genotype TH-GAL4 > 20xUAS-mCD8::GFP from control animals (A,A′) as well as from animals treated for 10 d with rotenone (B,B′) were analyzed using anti GFP immunohistochemistry. GFP-positive, dopamine containing neurons are shown (arrows) in two different Z-areas (A,A′,B,B′). (C) Exposure of rotenone for different periods induced climbing disability in a time dependent manner. The distance reached by animals climbing a vertical plane within 20 s is given. Shown are mean values of three independent experiments, ±SEM, n = 20 individuals for each group (P < 0.01, *p < 0.005). (D,E) Magnetic bead assisted isolation of dopamine containing cells using beads coated with anti-mCD8 antibodies. (D) Cell suspension of decollated neurons containing GFP-positive (green arrows) as well as non-labeled neurons (white arrows). (E) GFP- positive cells (green arrows) and magnetic beads (white arrow heads) after bead-assisted isolation devoid of non-labeled cells. SCIENTIFIC REPOrTS \| (2018) 8:2372 \| DOI:10.1038/s41598-018-20836-w 3 ntificreports/ Figure 2. Early transcriptional response to rotenone treatment of dopamine containing cells. GO analyses of genes that were upregulated (A) or downregulated (B) in dopamine containing cells in response to rotenone treatment. Clustering was performed with the DAVID program package68. Functional categories overrepresented in the lists of genes differentially expressed in rotenone treated flies identified by DAVID (Table 1, p < 0.05). (C) Fold changes for genes associated with the GO term regulation of apoptosis (D) Oxidative phosphorylation (E) neurotransmitter secretion (F) ageing. Error bars represent SEM. Damm: Death associated molecule related to Mch2, Dcp-1: Death caspase 1, BI-1: Bax Inhibitor 1, Jafrac2: Thioredoxin peroxidase 2, th: thread, PDCD-5: Programmed cell death 5, Hsp60D, out: Outsiders, sox15: Sox box protein 15, Prx2540-2: Peroxiredoxin 2540-2, Cyp6a2, Cyp12a5, Cyp12a4: Cytochrome p450-6a2, 12a4, 12a5, ti l S (T l) C VII C t h id b it VII CG6463 C C t i t i t i F 2 www.nature.com/scientificreports/ Figure 2. Early transcriptional response to rotenone treatment of dopamine containing cells. GO analyses of genes that were upregulated (A) or downregulated (B) in dopamine containing cells in response to rotenone treatment. Clustering was performed with the DAVID program package68. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Database identifier Functional category (Enriched GO terms) Number of changed genes p-value < 0.05 Upregulated genes GO:0032535 Regulation of cell size/Neurogenesis 13 0.022 GO:0042981 Apoptosis 10 0.030 GO:0007568 Ageing 10 0.030 KEGG_PATHWAY MAPK signaling pathway 6 KEGG_PATHWAY mTOR signaling pathway 4 KEGG_PATHWAY TGF beta signaling pathway 5 SM00034 CLECT C-type 6 0.040 GO:0007269 Cell-cell signaling/neurotransmitter secretion 15 0.040 GO:0000022 Motor activity 11 0.003 GO:0046146 Tetrahydrobiopterin metabolic process 4 0.012 Downregulated genes GO:0050877 Neurological system processes 20 0.014 KEGG_PATHWAY Wnt signaling pathway 6 GO:0010564 Regulation of cell cycle process 6 0.024 GO:0009310 Amine catabolic process 4 0.040 Table 1. Enriched functional categories for genes differentially regulated by rotenone. Table 1. Enriched functional categories for genes differentially regulated by rotenone. cell cycle were also enriched. Applying the DAVID program package to the data revealed additional GO terms as being overrepresented. Those genes with functions related to cellular transport were changed with the highest fre- quency (83 genes), followed by various others (Fig. 2A). Other important categories are related to stress response (14 genes), regulation of apoptosis (13 genes), protein folding (12 genes) and ageing (10 genes) (Fig. 2A). These results might be interpreted as a defense response towards a mild toxic offence given by rotenone. Likewise, a list of down-regulated genes was generated and clustered into 9 relevant functional categories (Fig. 2B). g g g g ( g ) Collectively, GO terms that were highly enriched in the combined cohorts of up- and down-regulated genes included regulation of apoptosis (Fig. 2C), oxidative phosphorylation (OXPHOS, Fig. 2D), neurotransmitter secretion (Fig. 2E) or ageing (Fig. 2F). Additional relevant clusters with high enrichment score and p < 0.05 and KEGG pathways filtered by DAVID are listed in Table 1 and Fig. S1, respectively. To validate the differential expression obtained using the microarray data, we performed qRT-PCR experiments with 3 informative genes, namely down-regulation of dat (Dopamine N-acetyltransferase) as well as the enhanced expression of calmodulin and of lethal2NC136 (Fig. S2). Pathway enrichment analysis. Since pathway analysis is anticipated to give more instructive data than single gene analysis28, further analyses focused on those pathways, whose activity was apparently deregulated. KEGG analysis revealed four pathways to be significantly altered, namely; the mitogen-activated protein kinases (MAPK/EGFR), the transforming growth factor beta (TGF-β), the target of rapamycin (Tor) and the Wnt signal- ing pathway. www.nature.com/scientificreports/ 3D). For the TGF-β signaling pathway, the expression level of its canonical target gene, Daughter against dpp (Dad) was analysed. Its expression level was sig- nificantly increased in rotenone treated samples (p < 0.05; Fig. 3E), indicative for an increased pathway activity. In order to evaluate the relevance of modulating the candidate signaling pathways, we employed the Gal4/ UAS-system. Using the TH-Gal4 driver line, this manipulation was restricted to DA containing cells only. We focussed on overexpression of armadillo (using UAS-arm) as the most important signalling pathway impaired by rotenone treatment. While the climbing performance index (relative proportion of animals that were able to cross a 2 cm threshold within 10 s) was identical between controls and those overexpressing armadillo were identical at the beginning of rotenone treatment, the decline in performance from day 10 onwards was dramatic while this was not the case for animals with ectopically increased armadillo expression in dopamine-producing cells only (Fig. 4). These differences were statistically highly significant. i y p p g p y y In order to evaluate the relevance of modulating the candidate signaling pathways, we employed the Gal4/ UAS-system. Using the TH-Gal4 driver line, this manipulation was restricted to DA containing cells only. We focussed on overexpression of armadillo (using UAS-arm) as the most important signalling pathway impaired by rotenone treatment. While the climbing performance index (relative proportion of animals that were able to cross a 2 cm threshold within 10 s) was identical between controls and those overexpressing armadillo were identical at the beginning of rotenone treatment, the decline in performance from day 10 onwards was dramatic while this was not the case for animals with ectopically increased armadillo expression in dopamine-producing cells only (Fig. 4). These differences were statistically highly significant. Results R The GOrilla analysis revealed a significant enrichment of the GO terms regulation of cell death and negative regu- lation of cell death (p values of 8.8 10−4 and 5.3 10−5, respectively). Moreover, terms such as negative regulation of SCIENTIFIC REPOrTS \| (2018) 8:2372 \| DOI:10.1038/s41598-018-20836-w 4 www.nature.com/scientificreports/ While activity of the first three pathways appear to be increased, that of the Wnt pathway appears to be reduced (Fig. 3, Table 1). Genes tightly associated with the MAPK/EGFR signaling are shown in Fig. 3A, while those associated with the Wnt pathway are shown in Fig. 3B. Expression levels of genes overrepresented in TGF-β and Tor signaling are shown in Table S1. In order to evaluate if the activity of the corresponding pathways were indeed induced or impaired, we analyzed either canonical pathway genes of these pathways or essential path- way constituents by qRT-PCR. For the MAPK/EGFR-pathway, expression of the canonical pathway gene spitz29 was upregulated several fold (p < 0.05; Fig. 3C). For Wnt signaling we analyzed armadillo, a central regulatory component of the Wnt signaling pathway (the β-catenin homolog). The relative expression level of armadillo in rotenone treated samples was significantly reduced (p < 0.01; Fig. 3D). For the TGF-β signaling pathway, the expression level of its canonical target gene, Daughter against dpp (Dad) was analysed. Its expression level was sig- nificantly increased in rotenone treated samples (p < 0.05; Fig. 3E), indicative for an increased pathway activity. Pathway enrichment analysis. Since pathway analysis is anticipated to give more instructive data than single gene analysis28, further analyses focused on those pathways, whose activity was apparently deregulated. KEGG analysis revealed four pathways to be significantly altered, namely; the mitogen-activated protein kinases (MAPK/EGFR), the transforming growth factor beta (TGF-β), the target of rapamycin (Tor) and the Wnt signal- ing pathway. While activity of the first three pathways appear to be increased, that of the Wnt pathway appears to be reduced (Fig. 3, Table 1). Genes tightly associated with the MAPK/EGFR signaling are shown in Fig. 3A, while those associated with the Wnt pathway are shown in Fig. 3B. Expression levels of genes overrepresented in TGF-β and Tor signaling are shown in Table S1. In order to evaluate if the activity of the corresponding pathways were indeed induced or impaired, we analyzed either canonical pathway genes of these pathways or essential path- way constituents by qRT-PCR. For the MAPK/EGFR-pathway, expression of the canonical pathway gene spitz29 was upregulated several fold (p < 0.05; Fig. 3C). For Wnt signaling we analyzed armadillo, a central regulatory component of the Wnt signaling pathway (the β-catenin homolog). The relative expression level of armadillo in rotenone treated samples was significantly reduced (p < 0.01; Fig. Discussionh Although Drosophila models of Parkinson’s disease recapitulate most aspects of the disease, this appears to be based on massive functional impairments of dopaminergic cells rather than on their death24. Thus, we analyzed in the current disease model a very early stage of cell pathology that later develops into the disease-associated functional impairments. We used a toxin-induced model of Parkinson’s disease to enhance the freedom of operation for genetic manipulation. Although we decided to utilize rotenone treatment, rotennone and paraquat models are equally well-suited for the induction of Parkinson’s disease symptoms in the fly24,31. The gene expression analysis carried out in this study is, to our knowledge, the first for rotenone-induced Parkinsonism in Drosophila that was focused on DA neurons. Prior studies of gene expression in Drosophila models of neurodegenerative diseases have been limited to studies of homogenates of brain tissue32,33. In con- trast, a comparable approach has been performed in mammals, namely in rats. Rotenone treatment followed by laser-dissection and transcriptome analyses of dopaminergic neurons revealed complex sets of regulated genes34. A closer comparison of the sets of genes that are regulated in dopamine-containing neurons of rats and flies revealed some surprising commonalities in their response characteristics to rotenone treatment. Regulated Gene Ontologies comprise those associated with cell death and cell cycle as well as those directly associated with gen- eral neuronal activities such as neurotransmitter release, which implies that the reaction types of rat and fly dopamine-containing neurons are surprisingly similar. In human brain tissues, the laser capture microdissection (LCM) technique has recently been employed for capturing only DA neurons for transcriptional profiling stud- ies35,36. The complex profile of genes with differentially regulated expression in response to rotenone treatment comprises some highly enriched pathways and gene ontologies. Presumably most relevant was the regulation of signaling pathways in these cells that are known to be highly relevant for cell survival and cell death, such as the MAPK/EGFR, TGF-β, Tor and Wnt pathways. be labeled specifically. Analyzing the early stage is of prime importance, because, at least in humans, clinically noticeable phenotypes occur after an estimated 70% of susceptible DA neurons in the Substantia nigra have already been destroyed30. Although Drosophila models of Parkinson’s disease recapitulate most aspects of the disease, this appears to be based on massive functional impairments of dopaminergic cells rather than on their death24. Discussionh The major aim of the current study was to provide new information about the molecular signatures associated with early phases of an induced Parkinson’s disease like phenotype that develop specifically in dopamine contain- ing neurons of the brain. In order to reach this ambitious goal, focusing exclusively on these dopamine producing cells being in an early stage was mandatory. Drosophila is ideally suited for this purpose as PD-like phenotypes can be induced easily, their progression can be quantified non-invasively and the dopamine-producing cells can SCIENTIFIC REPOrTS \| (2018) 8:2372 \| DOI:10.1038/s41598-018-20836-w 5 www.nature.com/scientificreports/ ntificreports/ Figure 3. Early modifications of signaling pathways in dopamine containing neurons in a rotenone induced PD model. (A) Shows up-regulated genes associated with MAPK/EGFR signaling pathway derived from the microarray data, and down-regulated genes associated with Wnt signaling pathway (B). qRT-PCR analysis of canonical target genes of the corresponding pathways (C–E). Spitz, a canonical target gene of the EGFR- pathway (C), Armadillo (arm) a central mediator of the wnt-pathway (D) and dad, a canonical TGF-β-pathway response gene (E). Ras85D: Ras Oncogene at 85D, Egfr: Epidermal growth factor receptor, RasGAP1: Ras GTPase activating protein 1, Rab14, Pk92B or DASK1: Protein kinase at 92B, drak: Death associated protein kinase related, skpA:skpA, CanA1: Calcineurin A1, smox: Smad on X, sgg: Shaggy: CycD: Cyclin D. Figure 3. Early modifications of signaling pathways in dopamine containing neurons in a rotenone induced PD model. (A) Shows up-regulated genes associated with MAPK/EGFR signaling pathway derived from the microarray data, and down-regulated genes associated with Wnt signaling pathway (B). qRT-PCR analysis of canonical target genes of the corresponding pathways (C–E). Spitz, a canonical target gene of the EGFR- pathway (C), Armadillo (arm) a central mediator of the wnt-pathway (D) and dad, a canonical TGF-β-pathway response gene (E). Ras85D: Ras Oncogene at 85D, Egfr: Epidermal growth factor receptor, RasGAP1: Ras GTPase activating protein 1, Rab14, Pk92B or DASK1: Protein kinase at 92B, drak: Death associated protein kinase related, skpA:skpA, CanA1: Calcineurin A1, smox: Smad on X, sgg: Shaggy: CycD: Cyclin D. be labeled specifically. Analyzing the early stage is of prime importance, because, at least in humans, clinically noticeable phenotypes occur after an estimated 70% of susceptible DA neurons in the Substantia nigra have already been destroyed30. Discussionh All animals were subjected to rotenone treatment and the climbing abilities of the experimental (blue) as well as of the control animals (red, background line for both insertions w1118, control = w1118) were analyzed. The distance reached by animals climbing a vertical plane within 10 s is given (N = 6 independent experiments, mean ± SEM, p < 0.001). Figure 4. Effect of armadillo overexpression in dopamine-containing cells in the rotenone induced PD model. Armadillo was specifically overexpressed by using TH-Gal4/UAS-arm animals. All animals were subjected to rotenone treatment and the climbing abilities of the experimental (blue) as well as of the control animals (red, background line for both insertions w1118, control = w1118) were analyzed. The distance reached by animals climbing a vertical plane within 10 s is given (N = 6 independent experiments, mean ± SEM, p < 0.001). controlling diverse cellular processes including tissue differentiation, neuronal survival, synaptogenesis and plasticity, as well as neurogenesis and neuroprotection9,37,38. Moreover, deregulated Wnt signaling is believed to be involved in various neuropathologies including Alzheimer’s disease, Schizophrenia and PD8,10. Canonical Wnt/β-catenin signaling appears to be highly important for controlling DA neuronal fate decision39. Remarkably, both GSK3-β inhibition and β-catenin stabilization increased commitment of neural precursors to develop into DA neurons40. Importantly, current studies imply that PD pathophysiology is associated with dysregulation of Wnt signaling8,9,14,41. Cantuti-Castelverti and colleagues reported a down-regulation of β-catenin levels in DA neurons of the Substantia nigra in PD patients36. In addition, proteins encoded by PARK genes, which were also shown to be involved in hereditary forms of PD, can modify Wnt pathway activity. LRRK2 (leucine-rich repeat kinase 2), which is associated with familial PD42 was shown to be connected to Wnt signaling8,13 and Parkin, an E3 ubiquitin ligase, regulates β-catenin protein levels in vivo14. Furthermore, gene expression profiling in progres- sively MPTP-lesioned macaques indicated down-regulation of β-catenin and dysregulation of key components of Wnt signaling28. Our results are consistent with these discussed findings, suggesting a general role of Wnt signaling in DA neuron welfare and PD development upon its impairment. Apparently, a certain level of Wnt signaling is necessary to guarantee survival of dopamine-containing neurons, especially in times of stress. Our rescue experiments support the view that downregulation of Wnt signaling is a key event in the neuropathology of PD and that amending this impairment improves the functionality and presumably also the survival of DA neurons at risk. Discussionh Thus, we analyzed in the current disease model a very early stage of cell pathology that later develops into the disease-associated functional impairments. We used a toxin-induced model of Parkinson’s disease to enhance the freedom of operation for genetic manipulation. Although we decided to utilize rotenone treatment, rotennone and paraquat models are equally well-suited for the induction of Parkinson’s disease symptoms in the fly24,31. The gene expression analysis carried out in this study is, to our knowledge, the first for rotenone-induced Parkinsonism in Drosophila that was focused on DA neurons. Prior studies of gene expression in Drosophila models of neurodegenerative diseases have been limited to studies of homogenates of brain tissue32,33. In con- trast, a comparable approach has been performed in mammals, namely in rats. Rotenone treatment followed by laser-dissection and transcriptome analyses of dopaminergic neurons revealed complex sets of regulated genes34. A closer comparison of the sets of genes that are regulated in dopamine-containing neurons of rats and flies revealed some surprising commonalities in their response characteristics to rotenone treatment. Regulated Gene Ontologies comprise those associated with cell death and cell cycle as well as those directly associated with gen- eral neuronal activities such as neurotransmitter release, which implies that the reaction types of rat and fly dopamine-containing neurons are surprisingly similar. In human brain tissues, the laser capture microdissection (LCM) technique has recently been employed for capturing only DA neurons for transcriptional profiling stud- ies35,36. The complex profile of genes with differentially regulated expression in response to rotenone treatment comprises some highly enriched pathways and gene ontologies. Presumably most relevant was the regulation of signaling pathways in these cells that are known to be highly relevant for cell survival and cell death, such as the MAPK/EGFR, TGF-β, Tor and Wnt pathways. β p y Of special interest in this study was the observed down-regulation of Wnt signaling in response to mild rote- none treatment in young adult flies. Armadillo, the Drosophila β-catenin acts as a transcription factor induc- ing expression of Wnt target genes. Wnt signaling is known throughout the animal kingdom to be involved in SCIENTIFIC REPOrTS \| (2018) 8:2372 \| DOI:10.1038/s41598-018-20836-w 6 www.nature.com/scientificreports/ Figure 4. Effect of armadillo overexpression in dopamine-containing cells in the rotenone induced PD model. Armadillo was specifically overexpressed by using TH-Gal4/UAS-arm animals. Discussionh Thus, interfering with Wnt signaling in DA containing neurons may represent a very promising and novel therapeutic strategy. V i il W i li MAPK i li i l i il ll d D hil l Very similar as Wnt signaling, MAPK signaling is evolutionarily well conserved. Drosophila melanogaster expresses all three subgroups of MAPKs: Rl (Rolled; ERK homolog), dJNK/Basket (Drosophila homolog of JNK), and dp38a and dp38b (Drosophila homologs of p38)43,44. Impairments of MAPK signaling pathways have been associated with many diseases including Alzheimer’s, PD and diverse types of cancer45. Strikingly, even at this early stage of disease development, expression of four genes associated with the MAPK signaling pathway was approximately 2-fold increased in rotenone treated flies. Moreover, expression of the Egfr, which is the central receptor in this signaling pathway, was also increased. Mice lacking the EGFR develop neurodegenerative diseases and die early46. In culture models, EGF stimulated neurite outgrowth, increased dopamine uptake and enhanced long-term survival in cultured dopaminergic neurons. Ectopic activation of ERK1/2 in rotenone rat models of PD, on the other hand, protected dopamine neurons from cell death47. In Drosophila, proper functioning of EGFR signaling has been shown to be essential for learning and memory48. The increased expression of EGFR and Ras protein coding genes in rotenone treated Drosophila suggests that these neurons launch defensive mechanisms due to stress given by rotenone. These findings support the view that EGFR signaling promotes cell survival also in the vulnerable DA neurons.h The third highly relevant signaling system, the TGF-β pathway controls a surplus of cellular processes in both developing and adult organisms49,50. When components of the TGF-β pathway are disrupted, several human dis- eases, including neurodegeneration and cancer arise50–52. There is increasing support for a role of TGF-β signaling in neuronal maintenance function and degeneration52–54. As most of the other pathways mentioned above, the target of rapamycin (TOR) signaling pathway is evolu- tionary conserved. It regulates cell proliferation, cell motility, cell survival, protein synthesis and transcription55,56. Nucleolar disruption and associated oxidative stress were demonstrated to suppress mTOR activity in DA neu- rons, thereby providing the basis for neuronal degeneration and the development of parkinsonism57. However, major key players of the pathway (TOR, Akt and elF4B) appeared to be up-regulated in flies treated with rotenone in the early phase, which is indicative for induction of survival mechanisms. SCIENTIFIC REPOrTS \| (2018) 8:2372 \| DOI:10.1038/s41598-018-20836-w Methods l i Twenty male adult flies were placed into a 17 cm long glass tube at a given time point. The flies were tapped to the bottom of the tube and let to climb the tube. After 20 s (or 10 s), a photo was taken in which most of the healthy flies were expected to have crossed the escape line at a height of 6 cm. The height/distance climbed by each fly was analyzed by using Image J. g For the recue experiments, we used the performance index as a quantitative measure60. In brief, animals (20 each) were analyzed at the indicated time points by tapping them and counting the number of animals that crossed a 2 cm line within 10 s. The performance index is calculated as follows: PI = 0.5 ((total number of ani- mals + number of animals above the line − number of animals below the line) divided through the total number of animals). In different types of experiments, we used heights achieved after 10 s or 20 s, depending on prelimi- nary experiments under the respective conditions. Tissue collection and analysis. Seventy heads of F1 generation male adult flies from the crossings between TH-Gal4 and 20xUAS-IVSmCD8::GFP were dissected in pre-chilled HL361 buffer. Dopaminergic neurons were sorted based on their mCD8:GFP expression by magnetic Dynabeads MyOne Streptavidin T1 (Invitrogen, Oslo, Norway) according to a previously described protocol62. Briefly, the tissue sample was vortexed for 1 sec, the supernatant was discarded and the procedure was repeated 3–4 times until the supernatant became clear. The heads were transferred to a pre-chilled 7 ml Kontes tissue grinder (Fisher Scientific, Leicestershire, UK), which was rinsed with 1% BSA in HL3 buffer in order to avoid the cells from sticking to the glass surface. About 4 ml of HL3 buffer were added to the tissue grinder and gently using a pestle, the tissues were given 30–32 douncing strokes. The solution was then triturated 5 times using the fire-polished glass pipette narrowed to approximately 50% of the standard tip diameter. The level of dissociation was assessed using a fluorescent microscope with a GFP filter (Axiovert S. 100, Zeiss, Jena, Germany). Then the solution was filtered through a 30 µm cell filter (Miltenyi Biotec, Bergisch Gladbach, Germany). The Dynabeads coupled to undiluted biotinylated rat anti-mouse CD8a antibody (eBioscience, Frankfurt, Germany), was then added to the filtrate and incubated for 1 hour on ice. Methods l i Fly strains and husbandry. Fly stocks were raised on standard cornmeal-agar medium at 25 °C under 12 h/12 h on/off light cycle. The following flies were used in the experiments described below: TH-Gal4 males (Bloomington Drosophila Stock Center, USA) were crossed to virgin females of either 20xUAS-IVSmCD8::GFP or UAS-arm (Bloomington Drosophila Stock Center, USA) to generate flies overexpressing GFP or armadillo in dopaminergic neurons, respectively. Toxin administration. Rotenone (Sigma Aldrich, Deisenhofen, Germany) was administered orally accord- ing to a previously described procedure23 with minor modifications. Briefly, 3-day- old flies from the crossing mentioned above were exposed to 0.5 mM rotenone in 10% glucose on blotting paper at 25 °C. A volume of 300 µl rotenone/glucose solution was added every 48 h to avoid desiccation. For the control group the same volume of 10% glucose only was added. g y In order to evaluate the relevance of selected signaling pathways for survival of dopamine containing neurons in the presence of rotenone, the Gal4/UAS system was employed utilizing the TH-Gal4 driver and effector lines used to ectopically overexpress relevant genes. Immunohistochemistry. Immunohistochemistry was performed as previously described58. Brains were dissected manually in Drosophila Ringer’s solution and immediately fixed in 4% paraformaldehyde in PBS for 30 min at room temperature. Subsequently, the samples were washed with PBST (0.3% Triton X-100 in PBS) and blocked in blocking-buffer (10% goat serum in PBST) for 30 min at room temperature, followed by incubation with the primary antibody (1:300 rabbit anti-GFP, Sigma-Aldrich, Taufkirchen, Germany) overnight at 4 °C with subsequent application of the secondary antibody (1:500 anti-rabbit DL488, Jackson ImmunoLabs, Suffolk, UK) for 3 h at room temperature. After washing, the brains were mounted on slides and images were obtained using a fluorescent microscope equipped with an apotome (Zeiss Axio Imager Z1, Göttingen, Germany). To quantify the effects of rotenone on cell numbers, fly brains of treated and non-treated animals (rotenone treatment for 10 days) were dissected and processed as described above. Z-stack images of rotenone treated and control fly brains were acquired using a. The number of GFP-labeled cells in the central brain (without optic lobes) was determined per brain hemisphere with the Fiji Cell Counter plugin, which facilitates cell counting of 3D images59. Behavioural testing-Climbing ability (negative geotaxis) assay. Locomotor ability of adult flies was tested by a negative geotaxis assay as described previously16 with minor modifications. Discussionh y Collectively, this study shows that the expression of several genes involved in the MAPK/EGFR, TGF-β and TOR signaling pathways were increased, presumably in order to launch a protective cellular program. The observed down-regulation of Wnt signaling on the other hand may reflect early signs of neurodegeneration. Thus, SCIENTIFIC REPOrTS \| (2018) 8:2372 \| DOI:10.1038/s41598-018-20836-w 7 www.nature.com/scientificreports/ increasing expression of either of the pathways mentioned above may increase survival of dopamine-containing neurons during disease progression, a hypothesis that is supported by the first experiments performed in this study focusing on enhancing Wnt signaling. increasing expression of either of the pathways mentioned above may increase survival of dopamine-containing neurons during disease progression, a hypothesis that is supported by the first experiments performed in this study focusing on enhancing Wnt signaling. Methods l i Following this, the tubes were placed on a MagnaRack (Invitrogen, Karlsuhe, Germany) for 2 min to pellet the beads along with GFP positive cells. The supernatant was removed and discarded. Then the cells were washed three times with ice cold HL3 buffer by putting the tubes on the magnet to remove non-specific cell binding. Cells bound to the coupled beads were resuspended in 30 µl of HL3 buffer and their purity and yield were controlled by taking 5 µl and observe it under the fluorescent microscope. Approximately, 8–10 GFP-positive cells were counted at one time point. Total RNA (from approx. 200 DA neurons) was isolated using the RNA NucleoSpin Tissue Kit (Macherey & Nagel, Düren, Germany). The RNA pellet was resolved in 10 µl RNase-free H2O and stored at −80 °C until analyses. 8 SCIENTIFIC REPOrTS \| (2018) 8:2372 \| DOI:10.1038/s41598-018-20836-w www.nature.com/scientificreports/ Microarray analysis. Microarray analyses were performed as described earlier63,64. Briefly, cDNA was synthesized by using PrimeScript RT (Takara Bio Europe, Saint Germain-en-Laye, France) according to the manufacturer’s protocol using a CapFinder approach in order to amplify the entire cDNA population65,66. The following primers were employed: CapFinderSp6rG (5′-CAG CGG CCG CAG ATT TAG GTG ACA CTA TAG A rGrGrG-3′) and OdT T7 I (5′-GAG AGA GGA TCC AAG TAC TAA TAC GAC TCA CTA TAG GGA GAT TTT TTT TTT TTT TTT TTT T G/A/C-3′). cDNA was amplified with OdT T7 II (5-GAG AGA GGA TCC AAG TAC TAA TAC GAC TCA CTA TAG G-3′) and Adaptor Sp6rG (5′-GAC GCC TGC AGG CGA TGA ATT TAG G-3′) and LA Taq polymerase. In vitro transcription of cDNA was performed with MEGAscript® T7 including aminoallyl-UTP and subsequently labeled with Alexa Fluor 647 or 555 (Life Technologies, Darmstadt, Germany) for treated or control sample, respectively. cRNA probes were hybridized to microarray slides (Canadian Drosophila Microarray Centre, Toronto, Canada) and scanned with a Gene Pix 4000B scanner equipped with the Gene Pix Pro 6.0 software (Axon Instruments, Science Products, Hofheim, Germany). Data normalization of the probe signal intensity levels across the arrays was performed with Acuity 4.1 (Axon Instruments, Science Products, Hofheim, Germany). A fold change of >1.5 of the mean signal intensity of a specific gene in at least 2 arrays out of three was considered as up-regulated and fold change <0.67 as down-regulated. References 1. Kalia, L. V. & Lang, A. E. Parkinson’s disease. Lancet 386, 896–912, https://doi.org/10.1016/S0140-6736(14)61393-3 (2015). 2. Pringsheim, T., Jette, N., Frolkis, A. & Steeves, T. D. The prevalence of Parkinson’s disease: a systematic review and meta-an 1. Kalia, L. V. & Lang, A. E. Parkinson’s disease. Lancet 386, 896–912, https://doi.org/10.1016/S0140-6736(14)61393-3 (2015). 2. Pringsheim, T., Jette, N., Frolkis, A. & Steeves, T. D. The prevalence of Parkinson’s disease: a systematic review and meta-analysis. Mov Disord 29, 1583–1590, https://doi.org/10.1002/mds.25945 (2014). 1. Kalia, L. V. & Lang, A. E. Parkinsons disease. 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SCIENTIFIC REPOrTS \| (2018) 8:2372 \| DOI:10.1038/s41598-018-20836-w SCIENTIFIC REPOrTS \| (2018) 8:2372 \| DOI:10.1038/s41598-018-20836-w 10 SCIENTIFIC REPOrTS \| (2018) 8:2372 \| DOI:10.1038/s41598-018-20836-w Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-20836-w.h Competing Interests: The authors declare that they have no competing interests. Competing Interests: The authors declare that they have no competing interests. ublisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional 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 Cre- ative Commons license, and indicate if changes were made. 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https://openalex.org/W4384305475	https://jurnal.fe.umi.ac.id/index.php/CSEJ/article/download/444/301	Indonesian	null	Pengaruh Keselamatan dan Kesehatan Kerja Terhadap Produktivitas Kerja Karyawan Bagian Engineering di PT. Industri Kapal Indonesia (IKI) Makassar	Center of Economic Students Journal	2,021	cc-by	4,139	Pengaruh Keselamatan dan Kesehatan Kerja Terhadap Produktivitas Kerja Karyawan Bagian Engineering di PT. Industri Kapal Indonesia (IKI) Makassar Almaudio Nefaldri Achmad1, Aryati Arfah2, La Mente3, Moh. Zulkifli Murfat4 almaudionefaldri@gmail.com1, aryati.arfah@umi.ac.id2, la.mente@umi.ac.id3, mohzulkifli.murfat@umi.ac.id4 Almaudio Nefaldri Achmad1, Aryati Arfah2, La Mente3, Moh. Zulkifli Murfat4 almaudionefaldri@gmail.com1, aryati.arfah@umi.ac.id2, la.mente@umi.ac.id3, mohzulkifli.murfat@umi.ac.id4 1Program Studi Manajemen, Fakultas Ekonomi dan Bisnis,Universitas Muslim Indonesia 2,3,4Fakultas Ekonomi dan Bisnis, Universitas Muslim Indonesia 1Program Studi Manajemen, Fakultas Ekonomi dan Bisnis,Universitas Muslim Indonesia 2,3,4Fakultas Ekonomi dan Bisnis, Universitas Muslim Indonesia Abstrak Penelitian ini bertujuan untuk menguji pengaruh keselamatan dan kesehatan kerja terhadap produktivitas kerja karyawan bagian engineering di Utara. Data dalam penelitian ini diperoleh dari masing-masing pegawai di PT. Industri Kapal Indonesia (IKI) Makassar yang bersedia menjadi responden. Penelitian ini menggunakan data primer dengan cara melakukan penelitian langsung dilapangan dengan memberikan kuesioner/lembar pertanyaan kepada 82 responden. Metode analisis data yang digunakan yaitu analisis regresi linear sederhana. Hasil penelitian menunjukkan bahwa terdapat pengaruh keselamatan dan kesehatan kerja terhadap produktivitas kerja karyawan pada bagian engineering di PT. Industri Kapal Indonesia (IKI) Makassar yang signifikan dan positif yang didasarkan perhitungan koefisien determinasi adalah sebesar 35,16%, sementara sisanya dipengaruhi oleh faktor lain yang tidak diteliti sebesar 64,84%. Kata kunci : Kesehatan dan Keselamatan Kerja, Produktivitas Kerja Center of Economic Student Journal Vol. 4 No. 3, Juli 2021 e-issn : 2621 - 8186 Center of Economic Student Journal Vol. 4 No. 3, Juli 2021 e-issn : 2621 - 8186 Center of Economic Student Journal 4(3) (2021) \| 215 Pendahuluan Dunia industri sekarang berkembang pesat termasuk di tanah air. Hal ini tentunya berdampak positif terhadap penyerapan tenaga kerja. Bisnis yang sarat akan persaingan sekarang ini menimbulkan berbagai cara bagi perusahaan untuk meningkatkan produktivitas. Salah satu cara yang dilakukan adalah upaya peningkatan produktivitas karyawan. Tidak dapat dipungkiri bahwa, teknologi mutakhir sangat lebih dari menunjang produktivitas, akan tetapi bagaimanapun juga motor penggerak teknologi tetaplah manusia (karyawan). Sumber daya manusia merupakan satu-satunya sumber daya yang memiliki akal, perasaan, keinginan, kemampuan, keterampilan, pengetahuan, dorongan, daya dan karya, satu-satunya sumber daya yang memiliki rasio, rasa dan karsa betapapun majunya teknologi berkembangnya informasi, tersedianya modal dan memadainya bahan namun jika tanpa SDM maka akan sulit bagi organisasi untuk mencapai tujuannya (Wahjono, 2016). Sumber daya manusia tak akan tergantikan. Mengembangkan tenaga kerja adalah tugas penting Manajemen Sumber Daya Manusia (MSDM) sebagaimana fungsi Manajemen SDM yaitu fungsi manajerial dan fungsi operasional. Ketika kita lebih menekankan pada fungsi operasional maka MSDM berfungsi pada pengadaan Center of Economic Student Journal 4(3) (2021) \| 215 Center of Economic Student Journal Vol. 4 No. 3, Juli 2021 e-issn : 2621 - 8186 tenaga kerja, pengembangan, kompensasi, integrasi, pemberhentian tenaga kerja, dan pemeliharaan. Fungsi pemeliharaan adalah memelihara apa yang telah dibentuk yaitu angkatan kerja yang efektif, semangat, dan kegairahan kerja. Fungsi ini dititik beratkan pada pemeliharaan fisik dan mental para karyawan melalui program Keselamatan dan Kesehatan Kerja Karyawan (K3). Sebagaimana yang dikemukakan oleh Gunara (2017) Pencegahan dan pemberantasan penyakit-penyakit dan kecelakaan-kecelakaan akibat kerja, pemeliharaan, peningkatan kesehatan dan gizi tenaga kerja, pemberantasan kelelahan kerja dan melipat gandakan kegairahan dan semangat kerja, perlindungan bagi masyarakat sekitar suatu perusahaan agar terhindar dari bahaya-bahaya yang mungkin ditimbulkan oleh produk-produk industri serta memberikan rasa aman kepada para karyawan. Oleh karena itu karyawan adalah aset penting perusahaan. Disinilah Manajemen Sumber Daya Manusia berperan penting. Sejauh mana perusahaan memberikan perhatian yang cukup terhadap kondisi kerja karyawan. Lebih mudah dalam bahasa sumber daya manusianya, Keselamatan dan Kesehatan Kerja Karyawan (K3). Terdapat berbagai sumber daya yang dibutuhkan dalam menjalankan suatu bisnis perusahaan, seperti modal, material dan mesin.Tidak terkecuali perusahaan juga membutuhkan sumber daya manusia, yaitu para karyawan.Karyawan yang diharapkan organisasi tentunya adalah karyawan yang dapat bekerja produktif, yaitu yang berkemampuan untuk menghasilkan produktivitas kerja yang optimal seperti yang direncanakan. Center of Economic Student Journal 4(3) (2021) \| 216 Pendahuluan Kesehatan dan Keselamatan Kerja suatu promosi dan pemeliharaan tertinggi tingkat fisik, mental, dan kesejahteraan sosial dimana terdapat pencegahan resiko mengurangi kecelakaan kerja, perlindungan pekerja dari risiko yang merugikan kesehatan, penempatan dan memelihara pekerja dalam lingkup kerja yang disesuaikan dengan peralatan fisiologis dan psikologis yang tidak membahayakan nyawa (Yuliandi & Ahmad, 2019). Keselamatan dan kesehatan kerja (K3) di tempat kerja merupakan salah satu aspek yang penting dan perlu mendapatkan perhatian dan penanganan serius, sebab apabila hal tersebut diabaikan dapat mengakibatkan kecelakaan bagi para pekerja yang berakibat pada menurunnya kualitas kerja yang dilakukan oleh para pekerja sehingga segala bentuk pekerjaan yang dilakukan akan mengalami hambatan seperti tenaga kerja yang diperlukan menjadi menurun (Simanjuntak & Abdullah, 2017). Faktor keselamatan kerja menjadi penting karena berkaitan dengan kinerja karyawan perusahaan. Semakin tersedianya fasilitas keselamatan kerja semakin sedikit kemungkinan terjadinya kecelakaan kerja. Terjadinya kecelakaan kerja berpengaruh buruk tidak hanya untuk karyawan yang mengalami kecelakaan sehingga kecelakaan kerja harus ditekan seminimal mungkin agar efek itu tak perlu terjadi. Menurut International Labour Organisation (ILO) atau Organisasi Buruh Internasional bahwa pada tahun 2019 sebanyak 2.78 juta jiwa yang meninggal akibat kecelakaan kerja dan 1.95 juta disebabkan oleh kecelakaan yang terjadi di lingkungan kerja. Dari kasus tersebut, 35-50% tenaga kerja di dunia kecelakaan kerja yang terjadi akibat dari paparan bahaya fisik, kimia dan biologi. Centers for Disease Control and Prevention (CDC) pada tahun 2019 yang mencatat bahwa adanya sejumlah 385.000 kasus kecelakaan kerja yang telah terjadi di Amerika Serikat karena adanya benda Center of Economic Student Journal 4(3) (2021) \| 216 Center of Economic Student Journal Vol. 4 No. 3, Juli 2021 e-issn : 2621 - 8186 tajam yang terkontaminasi darah pada tenaga kesehatan di Rumah Sakit Negara Amerika Serikat (International Labour Organisation, 2019). Badan Penyelenggara Jaminan Sosial (BPJS) Ketenagakerjaan menyebutkan angka kecelakaan kerja di sepanjang tahun 2017 sebanyak 147.000 kasus atau sebanyak 40.273 kasus setiap harinya. Dari jumlah itu, sebanyak 4.678 kasus (3.18%) berakibat kecacatan, dan 2575 kasus (1.75%) berakhir dengan kematian. Dari hasil data menunjukan, setiap hari ada 12 orang peserta BPJS Ketenagakerjaan mengalami kecacatan, dan 7 orang peserta meninggal dunia. Penyebab dari kecelakaan itu sendiri cukup beragam antara lain disebabkan oleh bencana alam, lingkungan atau peralatan yang tidak memenuhi syarat,dan perilaku yang tidak aman (BPJS Ketenagakerjaan, 2017). Setiap individu secara psikologis memiliki kebutuhan untuk merasa aman, terjamin dan dilindungi dari sesuatu yang dapat membahayakan. Pendahuluan Oleh karena itu, secara alamiah ia akan berusaha untuk tidak menempatkan dirinya pada posisi yang membahayakan dan bila ia tidak berhasil memperoleh kebutuhannya itu, maka jelas ia akan merasa tidak aman dan puas dalam bekerja, rasa tidak puas akan mempengaruhi semangat kerja dan tingkat kesehatan fisik maupun mental seorang tenaga kerja (Saputra, 2020). Program K3 merupakan salah satu usaha untuk melindungi karyawan ditempat kerja. Dengan terlindunginya karyawan dari was-was keselamatan dan kesehatan kerja, diharapkan akan dapat meningkatkan efisiensi perusahaan melalui peningkatan produktivitas karyawan (Prakoso et al., 2021). Berdasarkan penelitian terdahulu kepastian Penerapan Keselamatan dan Kesehatan Kerja Karyawan berdampak positif pada produktivitas kerja karyawan. Jika karyawan merasa puas dengan kondisi kerja mereka maka karyawan akan cenderung semangat dalam bekerja. Uraian di atas mendorong peneliti untuk melihat sejauh mana Penerapan Keselamatan dan Kesehatan Kerja Karyawan terutama pada bagian engineering di Kota Makassar mengingat bagian engineering merupakan salah satu bagian perusahaan yang berskala besar dan mempunyai karyawan yang banyak pula. Sehingga program keselamatan dan kesehatan kerja karyawan (K3) diperlukan dan memang ada di karyawan bagian engineering di PT Industri Kapal Indonesia (IKI) Makassar. Akan tetapi bagaimana dan sejauh mana pelaksanaan program tersebut terkait dengan produktivitas kerja karyawan, oleh karena itu berdasarkan fenomena tersebut peneliti tertarik mengangkat Judul Pengaruh Keselamatan dan Kesehatan Kerja Karyawan (K3) terhadap Produktivitas Kerja Karyawan bagian engineering di PT Industri Kapal Indonesia (IKI) Makassar. Oleh karena itu, penelitian ini bertujuan untuk menganalisis pengaruh keselamatan dan kesehatan kerja terhadap produktivitas kerja karyawan pada bagian engineering PT. Industri Kapal Indonesia (IKI) Makassar. Hipotesis penelitian adalah terdapat pengaruh positif dan signifikan antara keselamatan dan kesehatan kerja karyawan terhadap produktivitas kerja karyawan bagian engineering di PT. Industri Kapal Indonesia (IKI) Makassar. Center of Economic Student Journal 4(3) (2021) \| 217 Keselamatan Kerja dan Indikator Konsep tentang keselamatan kerja telah dikemukakan oleh para ahli dengan definisi dan batasan yang jelas. Menurut Gunara (2017), keselamatan kerja adalah suatu keadaan dalam lingkungan/ tempat kerja yang dapat menjamin secara maksimal keselamatan orang-orang yang berada di daerah/ tempat tersebut baik Center of Economic Student Journal Vol. 4 No. 3, Juli 2021 e-issn : 2621 - 8186 orang tersebut pegawai atau bukan pegawai dari organisasi kerja itu. Lingkup keselamatan kerja adalah keselamatan kerja bertalian dengan mesin, pesawat, alat kerja, bahan dan proses pengolahannya, tempat kerja dan lingkungannya serta cara-cara melakukan pekerjaan. Dalam Undang-undang nomor 14/tahun 1969 Pasal 9 dijelaskan bahwa “tiap tenaga kerja berhak mendapatkan perlindungan atau keselamatan, kesehatan, kesusilaan, pemeliharaan moral kerja serta perlakuan yang sesuai dengan martabat manusia dan moral agama”. Keselamatan dan kesehatan kerja adalah pengawasan terhadap orang, mesin, material, dan metode yang mencakup lingkungan kerja agar pekerja tidak mengalami cidera (Megawati, 2018). Menurut Suma’mur (Hidayatullah dan Tjahjawati, 2018) keselamatan kerja merupakan suatu keadaan terhindar dari bahaya saat melakukan kerja. Keselamatan kerja adalah keselamatan yang berhubungan dengan mesin, pesawat pengangkat, alat kerja, bahan dan proses pengolahannya, tempat kerja dan lingkungannya serta cara-cara melakukan pekerjaan. Menurut Simanjuntak keselamatan kerja dapat diartikan sebagai kondisi yang bebas dari risiko kecelakaan atau kerusakan atau dengan kata lain risiko yang relatif sangat kecil dibawah tingkat tertentu. Mangkunegara (2013: 196) mengatakan bahwa “keselamatan kerja adalah pengawasan terhadap orang, mesin, material, dan metode yang mencakup lingkungan kerja agar supaya pekerja tidak mengalami cidera.” Sementara Mirwan (2015:543) mengatakan bahwa “keselamatan kerja adalah kondisi dimana para pekerja selamat, tidak mengalami kecelakaan dalam melaksanakan tugas dan pekerjaanya. Dengan demikian, pelaksanaan pekerjaan berlangsung secara normal tidak terganggu oleh kecelakaan kerja, tenaga kerja dapat menciptakan kinerja yang direnacakan. Agar hal tersebut dapat tercipta perusahaan/ organisasi perlu melakukan manajemen keselamatan kerja yang merupakan bagian integrasi dari manajemen perusahaan/ organisasi. Keselamatan kerja menurut Prakoso, et.al (2021) menunjuk kondisi aman atau selamat dari penderitaan, kerusakan atau kerugian ditempat kerja. Risiko keselamatan merupakan aspek-aspek dari lingkungan kerja yang dapat menyebabkan kebakaran, ketakutan aliran listrik, terpotong, luka memar, keseleo, patah tulang, kerugian alat tubuh, penglihatan dan pendengaran. Semua itu sering dihubungkan dengan perlengkapan perusahaan atau lingkungan fisik dan mencakup tugas-tugas kerja yang membutuhkan pemeliharaan dan latihan. Berdasarkan pengertian keselamatan kerja di atas, maka dapat disimpulkan bahwa Keselamatan kerja adalah upaya untuk mengurangi kemungkinan terjadinya kecelakaan saat melakukan pekerjaan atau perlindungan dari kecelakaan dan cedera akibat suatu pekerjaan. Keselamatan Kerja dan Indikator Mangkunegara (2013:199) menyatakan bahwa indikator keselamatan kerja, yaitu: (a) pemakaian peralatan kerja / alat pelindung diri, (b) beban kerja, (c) peraturan keselamatan kerja, (c) komunikasi dan dukungan, dan (d) pelatihan dan keselamatan kerja. Center of Economic Student Journal 4(3) (2021) \| 218 Kesehatan Kerja dan Indikator Pengertian kesehatan kerja telah dikemukakan sejumlah ahli. Menurut Mirwan (2015:543), kesehatan kerja adalah “penerapan ilmu kesehatan/ kedokteran di bidang ketenagakerjaan yang bertujuan untuk mencegah penyakit yang timbul Center of Economic Student Journal 4(3) (2021) \| 218 Center of Economic Student Journal Vol. 4 No. 3, Juli 2021 e-issn : 2621 - 8186 akibat kerja dan mempertahankan dan meningkatkan kesehatan para pekerja/buruh untuk meningkatkan kinerja mereka. Kesehatan kerja berkaitan dengan penyakit yang diderita oleh seorang pekerja sebagai akibat aktivitas melaksanakan pekerjaanya.” Kecelakaan kerja seperti terjatuh, terbakar karena melaksanakan pekerjaan bukan merupakan penyakit kerja (occopational disease) tapi merupakan kecelakaan kerja. Penyakit kerja lebih diderita oleh para pekerja dan kurang nyata pada masyarakat umum. Misalnya, seorang buruh yang tugasnya memasang asbes dalam bangunan dapat terserang kanker paru-paru, disini kanker paru-paru merupakan penyakit kerja. Akan tetapi, ada orang yang terjangkit kanker paru-paru Karena perokok berat; dalam kasus kedua tersebut kanker paru-paru bukan penyakit kerja. Tenaga kerja harus dilindungi dari resiko pekerjaan seperti risiko fisik (ergonomic, bising, panas, dingin, getaran, radiasi, dan debu), resiko kimia, biologi (radiasi dan terkontaminasi bahan kimia dan kuman) serta stress karena beban kerja. Mangkunegara (2013:210) Kesehatan kerja adalah kondisi yang bebas dari gangguan fisik, mental emosi, atau rasa sakit yang disebabkan oleh lingkungan kerja. Kesehatan kerja adalah upaya dari perusahaan, pemerintah dan para pekerja/buruh sendiri untuk mencegah para pekerja/buruh mendapatkan penyakit karena melaksanakan pekerjaanya. Sedangkan kesehatan kerja menurut Prakoso, et.all (2021) menunjuk pada kondisi yang bebas dari gangguan fisik, mental, emosi atau rasa sakit yang disebabkan oleh lingkungan kerja. Risiko kesehatan merupakan faktor-faktor dalam lingkungan kerja yang bekerja melebihi periode waktu yang ditentukan lingkungan yang dapat membuat stress emosi atau gangguan fisik. Undang-undang Nomor 23 Tahun 1992, Pasal 23 tentang Kesehatan disebutkan bahwa Kesehatan kerja diselenggarakan untuk mewujudkan produktivitas kerja secara optimal yang meliputi pelayanan kesehatan pencegahan penyakit akibat kerja. Kesehatan Kerja adalah adanya jaminan kesehatan pada saat melakukan pekerjaan. Yang bertujuan untuk peningkatan dan pemeliharaan derajat kesehatan fisik, mental dan sosial yang setinggi-tingginya bagi pekerja di semua jenis pekerjaan, pencegahan terhadap gangguan kesehatan pekerja yang disebabkan oleh kondisi pekerjaan; perlindungan bagi pekerja dalam pekerjaannya dari resiko akibat factor yang merugikan kesehatan; dan penempatan serta pemeliharaan pekerja dalam suatu lingkungan kerja yang disesuaikan dengan kondisi fisiologi dan psikologisnya. Indikator kesehatan kerja dapat diukur dengan melihat dan mengkaji pandangan ahli. Kesehatan Kerja dan Indikator Menurut Mangkunegara (2013: 221) bahwa “pengukuran kesehatan kerja dapat dilihat melalui: (1) lingkungan kerja fisik; (2) sarana dan pelayanan kesehatan; (3) sarana rekreasi; dan (4) peraturan kesehatan kerja.” Center of Economic Student Journal 4(3) (2021) \| 219 Produktivitas Kerja dan Indikator Produktivitas kerja berasal dari kata produktif artinya segala kegiatan yang menimbulkan kegunaan (utility). Jika seseorang bekerja ada hasilnya, maka dikatakan produktif. Tetapi kalau menganggur,disebut tidak produktif, tidak menambah nilai guna bagi masyarakat. Orang-orang produktif dikatakan memiliki produktivitas yang tinggi. Produktivitas tidak saja diukur dari kuantitas (jumlah) hasil yang dicapai seseorang, tapi juga oleh mutu (kualitas) pekerjaan yang semakin baik. Makin baik mutu pekerjaannya, maka makin tinggi produktivitas kerjanya (Muayyad dan Gawi, 2017). Produktivitas menurut Katarina dan Kusumayadi (2021) dapat diartikan sebagai rasio output terhadap input. Input ini bisa mencakup biaya produksi Center of Economic Student Journal Vol. 4 No. 3, Juli 2021 e-issn : 2621 - 8186 Center of Economic Student Journal Vol. 4 No. 3, Juli 2021 e-issn : 2621 - 8186 Center of Economic Student Journal Vol. 4 No. 3, Juli 2021 e-issn : 2621 - 8186 dan biaya peralatan. Sedangkan output bisa terdiri dari penjualan, pendapatan, market share, dan kerusakan. Produktivitas merupakan ukuran atau standar yang dapat digunakan untuk melakukan perbandingan antara input atau modal yang telah dikeluarkan dengan hasil yang diperoleh. Semua perusahaan atau organisasi menghasilkan puluhan ribu jenis produk barang dan jasa yang diperlukan oleh masyarakat untuk memenuhi kebutuhan mereka. Dari kata produk, muncul konsep produksi, produktivitas, proses produksi, kuantitas dan kualitas produksi. Istilah produktivitas adalah rasio keluaran terhadap masukan yang mengukur efektivitas yang efisiensi dari produksi (Katarina dan Kusumayadi, 2021). Pengukuran produktivitas kerja perlu dilakukan untuk mengetahui produktivitas kerja dari setiap karyawan. Pengukuran produktivitas tenaga kerja menurut sistem pemasukan fisik per orang atau per jam kerja orang ialah diterima secara luas, dengan menggunakan metode pengukuran waktu tenaga kerja (jam, hari atau tahun). Pengukuran diubah ke dalam unit-unit pekerja yang diartikan sebagai jumlah kerja yang dapat dilakukan dalam satu jam oleh pekerja yang bekerja menurut pelaksanakan standar (Sinungan, 2013: 262) Menurut Simamora (2014: 612) faktor- faktor yang digunakan dalam pengukuran produktivitas kerja meliputi : a. Kuantitas kerja adalah merupakan suatu hasil yang dicapai oleh karyawan dalam jumlah tertentu dengan perbandingan standar ada atau ditetapkan oleh perusahan. a. Kuantitas kerja adalah merupakan suatu hasil yang dicapai oleh karyawan dalam jumlah tertentu dengan perbandingan standar ada atau ditetapkan oleh perusahan. b. Kualitas kerja adalah merupakan suatu standar hasil yang berkaitan dengan mutu dari suatu produk yang dihasilkan oleh karyawan dalam hal ini merupakan suatu kemampuan karyawan dalam menyelesaikan pekerjaan secara teknis dengan perbandingan standar yang ditetapkan oleh perusahaan. c. Produktivitas Kerja dan Indikator Ketepatan waktu merupakan tingkat suatu aktivitas diselesaikan pada awal waktu yang ditentukan, dilihat dari sudut koordinasi dengan hasil output serta memaksimalkan waktu yang tersedia untuk aktivitas lain.Ketepatan waktu diukur dari persepsi karyawan terhadap suatu aktivitas yang disediakan di awal waktu sampai menjadi output. Center of Economic Student Journal 4(3) (2021) \| 220 Metode Penelitian Populasi adalah sekelompok atau kumpulan dari objek atau objek yang akan digeneralisasikan dari hasil penelitian. Populasi penelitian adalah seluruh karyawan bagian engineering yang berjumlah 478 orang. Penulis memfokuskan pada lokasi penelitian di di PT Industri Kapal Indonesia (IKI) Makassar dengan waktu penelitian selama 2 bulan (Mei-Juni) tahun 2021. Pengambilan sampel secara purposive sampling dengan pertimbangan jumlah populasi yang besar dengan menggunakan rumus Slovin, sehingga ditetapkan jumlah sampel sebanyak 82 responden. Penelitian ini menggunakan dua variabel, yaitu: keselamatan dan kesehatan kerja (X) dan produktivitas kerja karyawan bagian engineering di PT. Industri Kapal Indonesia (IKI) Makassar. Jenis data dalam penelitian ini adalah data primer, yaitu data yang diperoleh melalui hasil penyebaran kuesioner kepada responden. Data sekunder, yaitu data pendukung, biasanya dapat diperoleh dari literatur, bahan pustaka, dan dokumen Center of Economic Student Journal 4(3) (2021) \| 220 Center of Economic Student Journal 4(3) (2021) \| 220 Center of Economic Student Journal Vol. 4 No. 3, Juli 2021 e-issn : 2621 - 8186 perusahaan yang berkaitan dengan masalah yang akan diteliti. Teknik pengumpulan data menggunakan observasi, angket (kuesioner) dan dokumentasi. Analisis data menggunakan statistik deskriptif dengan analisis regresi linier berganda dengan tingkat signifikansi 5% yang sebelumnya mensyaratkan uji prasyarat analisis dan uji asumsi klasik. Analisis Regresi Linier Sederhana Analisis persamaan regresi linear sederhan dapat disajikan pada tabel berikut ini : nalisis persamaan regresi linear sederhan dapat disajikan pada tabel berikut Tabel 1 Regresi Linear Sederhana Coefficientsa Model Unstandardized Coefficients Standardized Coefficients t Sig. B Std. Error Beta 1 (Constant) 13.846 2.749 5.036 <,001 Keselamatan_ Kesehatan_Kerja .493 .075 .593 6.594 <,001 a. Dependent Variable: Produktivitas_Kerja_Karyawan Sumber : Data Hasil Olahan SPSS 24, 2021 Model persamaan regresinya adalah Y= β+ β1X Y= 13,646 + 0.493X Berdasarkan persamaan tersebut, maka dapat digambarkan sebagai berikut: Tabel 1 Regresi Linear Sederhana Coefficientsa Model Unstandardized Coefficients Standardized Coefficients t Sig. B Std. Error Beta 1 (Constant) 13.846 2.749 5.036 <,001 Keselamatan_ Kesehatan_Kerja .493 .075 .593 6.594 <,001 a. Dependent Variable: Produktivitas_Kerja_Karyawan Sumber : Data Hasil Olahan SPSS 24, 2021 Model persamaan regresinya adalah Y= β+ β1X Y= 13,646 + 0.493X Berdasarkan persamaan tersebut, maka dapat digambarkan sebagai berikut: Tabel 1 Regresi Linear Sederhana Y= β+ β1X Y= 13,646 + 0.493X erdasarkan persamaan tersebut, maka dapat digambarkan sebagai berikut: Berdasarkan persamaan tersebut, maka dapat digambarkan sebagai berikut: Nilai konstanta (α) sebesar 13,846 menunjukkan bahwa jika nilai variabel bebas (kesehatan dan keselamatan kerja) bernilai 0 maka berbanding lurus dengan nilai variabel terikat (produktivitas kerja karyawan bagian engineering) yaitu 13,846. Nilai koefisien X (b) sebesar 0,493 ini berarti variabel produktivitas kerja karyawan bagian engineering berpengaruh positif terhadap variabel kesehatan dan keselamatan kerja, atau dengan kata lain jika variabel kesehatan dan keselamatan kerja ditingkatkan sebesar satu satuan, maka variabel produktivitas kerja bagian engineering sebesar 0,493. Uji Prasyarat Analisis (Validitas dan Reliabilitas) Hasil uji validitas dan reliabilitas pertanyaan angket, hasil uji validitas semua item pertanyaan dinyatakan valid karena diperoleh nilai Pearson Correlation lebih signifikan dari nilai rtabel sebesar 0,220. Kemudian untuk uji reliabilitas, nilai Cronbach's Alpha lebih besar dari batas 0,60 untuk memenuhi persyaratan reliabilitas. Center of Economic Student Journal 4(3) (2021) \| 221 Koefisien Determinasi R2(RSquare) Perhitungan koefisien determinasi dilakukan untuk menentukan seberapa besar pengaruh keselamatan dan kesehatan kerja terhadap produktivitas kerja karyawan bagian engineering, sebagaimana disajikan pada tabel sebagai berikut: Center of Economic Student Journal 4(3) (2021) \| 221 Center of Economic Student Journal 4(3) (2021) \| 221 Tabel 2 Koefisien Determinasi Model Summaryb Model R R Square Adjusted R Square Std. Error of the Estimate 1 .593a .352 .344 4.537 a. Predictors: (Constant), Keselamatan_Kesehatan_Kerja b. Dependent Variable: Produktivitas_Kerja_Karyawan Sumber : Data Hasil Olahan SPSS 24, 2021 Tabel 2 Koefisien Determinasi Sumber : Data Hasil Olahan SPSS 24, 2021 Berdasarkan tabel 2, dapat dilakukan perhitungan koefisien determinasi dengan rumus: KD = r2 x 100% = (0.593)2 x100% = 35,20% Berdasarkan hasil perhitungan koefisien determinasi, dapat dijelaskan bahwa pelaksanaan keselamatan dan kesehatan kerja mempunyai pengaruh sebesar 35,16% terhadap produktivitas kerja karyawan bagian engineering pada PT. Industri Kapal Indonesia (IKI) Makassar, sementara sisanya dipengaruhi oleh faktor lain yang tidak diteliti sebesar 64,84%. Faktor-faktor yang mempengaruhi produktivitas kerja adalah pelatihan, mental dan kemampuan fisik karyawan, hubungan antara atasan dan bawahan. Oleh karena itu, 64,84% dipengaruhi oleh mental dan kemampuan fisik karyawan dan hubungan antara atasan. Hasil dari program keselamatan dan kesehatan kerja pada PT. Industri Kapal Indonesia (IKI) Makassar dapat dikatakan baik. Hal ini didasarkan pada tanggapan responden karena nilai rata-rata dari keseluruhan pernyataan sebesar 3,62 yang berarti berada pada interval 3,40-4,19. Menurut Jackson, dkk (2011: 267), keselamatan dan kesehatan kerja mengacu pada kondisi fisiologis-fisikal dan psikologis pekerja yang merupakan hasil dari lingkungan yang diberikan oleh perusahaan. Jika suatu perusahaan melakukan pengukuran keamanan dan kesehatan yang efektif, maka semakin sedikit pegawai yang akan mengalami dampak penyakit jangka pendek atau jangka panjang akibat bekerja di perusahaan tersebut. Adapun hasil penelitian produktivitas kerja karyawan pada bagian engineering di PT. Industri Kapal Indonesia (IKI) Makassar dapat dikatakan baik berdasarkan tanggapan responden, karena nilai rata-rata dari keseluruhan pernyataan adalah sebesar 3,96 atau berada pada interval 3,40 - 4,19. Hal ini menunjukkan bahwa produktivitas kerja merupakan aspek penting bagi perusahaan. Hal ini sejalan dengan pandangan Gomes (2003: 159) bahwa produktivitas kerja merupakan suatu aspek yang penting bagi perusahaan karena apabila tenaga kerja dalam perusahaan mempunyai kerja yang tinggi, maka perusahaan akan memperoleh keuntungan dan hidup perusahaan akan terjamin. Usaha peningkatan produktivitas harus direncanakan secara baik dan sistematis, sehingga berhasil apabila diaplikasikan ke dalam suatu perusahaan.” Pengaruh keselamatan dan kesehatan kerja terhadap produktivitas kerja karyawan pada bagian engineering di PT. Center of Economic Student Journal 4(3) (2021) \| 222 Koefisien Determinasi R2(RSquare) Industri Kapal Indonesia (IKI) Makassar Center of Economic Student Journal 4(3) (2021) \| 222 Center of Economic Student Journal Vol. 4 No. 3, Juli 2021 e-issn : 2621 - 8186 didasarkan pada perhitungan koefisien determinasi sebesar 35,16%, sementara sisanya dipengaruhi oleh faktor lain yang tidak diteliti sebesar 64,84%. Dengan demikian, dapat disimpulkan bahwa pengaruh keselamatan dan kesehatan kerja terhadap produktivitas kerja karyawan pada bagian engineering di PT. Industri Kapal Indonesia (IKI) Makassar signifikan. Dari hasil penelitian ini diharapkan dapat dimanfaatkan bagi dunia kerja agar dapat membantu pemilik perusahaan melihat bagaimana pengaruh keselamatan dan kesehatan kerja terhadap produktivitas kerja karyawan terutama engineering. Perusahaan dapat melihat aspek ini sebagai pemikiran bagi kemajuan perusahaan dalam menghadapi persaingan yang semakin tajam. Simpulan Hasil penelitian disimpulkan bahwa terdapat pengaruh keselamatan dan kesehatan kerja terhadap produktivitas kerja karyawan pada bagian engineering di PT. Industri Kapal Indonesia (IKI) Makassar yang signifikan dan positif. Oleh karena itu, saran yang diberikan peneliti, yaitu (1) program keselamatan dan kesehatan kerja harus tetap dipertahankan,bahkan ditingkatkan sebab memiliki pengaruh yang signifikan terhadap produktivitas kerja. (2) pihak manajemen agar selalu memperhatikan pengelolaan dan pemeliharaan alat-alat produksi agar keselamatan dan kesehatan karyawan lebih terjamin, dan (3) kesadaran karyawan akan keselamatan diri dan lingkungan harus selalu ditingkatkan dengan cara memberikan apresiasi kepada karyawan yang berkontribusi lebih terhadap pelaksanaan keselamatan dan kesehatan kerja. Center of Economic Student Journal Vol. 4 No. 3, Juli 2021 e-issn : 2621 - 8186 Referensi BPJS Ketenagakerjaan. 2017. Laporan Teknis Kajian Aktuaria tentang Reformasi ( BPJS ) Ketenagakerjaan Indonesia Laporan Teknis Kajian Aktuaria tentang Reformasi Badan Penyelenggara Jaminan Sosial (BPJS) Ketenagakerjaan Indonesia. 1–105. http://hmkwetotm8.pdf-world.net/dl2.php?id=59430333&h= 0bbfae53fb6a38d06c060d6d1db08358&u=cache&ext=pdf&n=Bpjs ketenagakerjaan indonesia Gunara, S. 2017. Buku Pedoman Pelaksanaan Keselamatan dan Kesehatan Kerja. Occupational Health and Safety Guidebook, 1, 1–152. http://scbd.com/assets/uploads/files/1527233584SCBD_Buku_ Pedoman_Pelaksanaan_Keselamatan_dan_Kesehatan_Kerja_BP2K3_LR.compr essed-ilovepdf-compressed.pdf Hidayatullah, A., & Tjahjawati, S. S. 2018. Pengaruh Keselamatan dan Kesehatan Kerja Terhadap Produktivitas Kerja Karyawan. Jurnal Riset Bisnis Dan Investasi, 3(2), 104. https://doi.org/10.35697 /jrbi.v3i2.938 International Labour Organisation. 2019. Evolution of and prospects for women’s labour participation in Latin America. 21. Katarina, & Firmansyah Kusumayadi. 2021. Pengaruh Motivasi Kerja dan Disiplin Kerja Terhadap Produktivitas Kerja Karyawan pada CV.Makmur Jaya Abadi. Jurnal Inovasi Penelitian, 2(1), 199–204. ( ) Megawati, Y. 2018. Konsep Kesehatan dan Keselamatan Kerja di Rumah Sakit. Jurnal Kesehatan dan Keselamatan Kerja. Mangkunegara, A.A.P. 2013. Manajemen Sumber Daya Perusahaan. Remaja RosdaKarya, Bandung. Mirwan. 2015. Manajemen Sumber Daya Manusia Indonesia. PT Raja Grafindo Persada, Jakarta. Center of Economic Student Journal 4(3) (2021) \| 223 Center of Economic Student Journal Vol. 4 No. 3, Juli 2021 e-issn : 2621 - 8186 Muayyad, D. M., & Gawi, A. I. O. 2017. Pengaruh Kepuasan Kerja Terhadap Produktivitas Kerja Pegawai Bank Syariah X Kantor Wilayah II. Jurnal Manajemen dan Pemasaran Jasa, 9(1), 75. https://doi.org/ 10.25105/jmpj.v9i1.1396 Prakoso, S., Maulani, M., Nugrahanti, A., Samura, L., & Irham, S. 2021. Sosialisasi Program Keselamatan dan Kesehatan Kerja Bagi Karyawan CV Rumah Kampung Sawangan, Depok, Jawa Barat. Jurnal Abdi Masyarakat Indonesia, 3(1), 40.https://doi.org/10.25105/ jamin.v3i1.7800 Saputra, Rizky. 2020. Pengaruh Kompensasi, Keselamatan dan Kesehatan Kerja (K3) Serta Kepemimpinan Terhadap Produktivitas Kerja Karyawan pada PT. Indofood Sukses Makmur, Tbk Cabang Palembang. In Fakultas Ekonomi, Universitas Tridinanti (Vol. 5, Issue 1). Simanjuntak, R. A., & Abdullah, R. 2017. Tinjauan Sistem dan Kinerja Manajemen Keselamatan & Kesehatan Kerja Tambang Bawah Tanah CV. Tahiti Coal, Talawi, Sawahlunto, Sumatera Barat. Jurnal Bina Tambang, 3(4), 1536–1545. no, S. I. 2016. Manajemen sumber daya (Issue September 2015). Salemba mpat, Jakarta Yuliandi, C. D., & Ahman, E. 2019. Penerapan Keselamatan dan Kesehatan Kerja (K3) di Lingkungan Kerja Balai Inseminasi Buatan (BIB) Lembang. 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https://openalex.org/W2464518265	https://link.springer.com/content/pdf/10.1007/s00384-016-2621-7.pdf	English	null	Enhanced recovery care after colorectal surgery in elderly patients. Compliance and outcomes of a multicenter study from the Spanish working group on ERAS	International journal of colorectal disease	2,016	cc-by	5,362	Abstract Purpose ERAS (enhanced recovery after surgery) programs have proven to reduce morbidity and hospital stay in colorec- tal surgery. However, the feasibility of these programs in el- derly patients has been questioned. The aim of this study is to assess the implementation and outcomes of an ERAS program for colorectal cancer in elderly patients. Conclusions ERAS after colorectal surgery in elderly patients presents as safe and feasible based on good reported outcomes of compliance rates, complications, readmissions, and needs for reoperation. Methods This is a multicenter observational study of a cohort of elderly patients undergoing colorectal surgery within an ERAS program. A total of 188 consecutive patients over 70 years who underwent elective colorectal surgery within an ERAS program at three institutions during a 2-year period were included. The compliance with the ERAS protocol inter- ventions was measure. Complications were evaluated accord- ing to Clavien-Dindo classification. Data on length of stay and readmission rates were analyzed. Keywords ERAS . Enhanced . Colorectal cancer . Elderly Keywords ERAS . Enhanced . Colorectal cancer . Elderly Enhanced recovery care after colorectal surgery in elderly patients. Compliance and outcomes of a multicenter study from the Spanish working group on ERAS Santiago Gonzalez-Ayora1 & Carlos Pastor1 & Hector Guadalajara1 & Jose Manuel Ramirez2 & Pablo Royo2 & Elizabeth Redondo2 & Antonio Arroyo3 & Pedro Moya3 & Damian Garcia-Olmo1 Accepted: 20 June 2016 /Published online: 4 July 2016 p y # The Author(s) 2016. This article is published with open access at Springerlink.com # The Author(s) 2016. This article is published with open access at Springerlink.com 13 % had major complications; of them, 8 % patients were reoperated. The readmission rate was 6.4 %. Conclusions ERAS after colorectal surgery in elderly patients presents as safe and feasible based on good reported outcomes of compliance rates, complications, readmissions, and needs for reoperation. 13 % had major complications; of them, 8 % patients were reoperated. The readmission rate was 6.4 %. 13 % had major complications; of them, 8 % patients were reoperated. The readmission rate was 6.4 %. Int J Colorectal Dis (2016) 31:1625–1631 DOI 10.1007/s00384-016-2621-7 Int J Colorectal Dis (2016) 31:1625–1631 DOI 10.1007/s00384-016-2621-7 ORIGINAL ARTICLE 1 Department of General Surgery, Division of Colorectal Surgery, Fundacion Jimenez-Diaz, Reyes Catolicos Ave #2, 28040 Madrid, Spain 3 Department of General Surgery, Division of Colorectal Surgery, Hospital General Universitario, Elche, Spain * Santiago Gonzalez-Ayora sgayora@fjd.es 2 Department of General Surgery, Division of Colorectal Surgery, Hospital Clinico Universitario, Zaragoza, Spain Introduction Over the last decade, the enhanced recovery after surgery (ERAS) programs have generated a true revolution in colorec- tal surgery. This revolution has been compared to the innova- tive concept of total mesorectal excision for rectal surgery [1] or to that of the adoption of laparoscopic surgery as the gold standard in colon surgery [2, 3]. Results Early intake and early mobilization were the most successfully carried out interventions. There was a global compliance rate of 56 % of patients for whom compliance was achieved with all measured interventions. The median hospital length of stay was 6 days. Almost 60 % of patients had no complications, 24 % had minor complications while Initially proposed by Professor Henrik Khelet from Denmark, ERAS is a multidisciplinary set of care interven- tions in patients with the goal to obtain a comprehensive re- covery after any surgical intervention [4, 5]. Focused on co- lorectal cancer surgery, there exist to date strong evidence demonstrating that adhesion to ERAS protocols can minimize morbidity by decreasing secondary complications while also being cost-effective in shortening the length of hospital stay (LOS) [6–11]. Along with all the benefits of ERAS, it is known that there are significant limitations in implementing these protocols, due to the fact that compliance of all interven- tions may be difficult to achieve by each patient, which may worsen end results [12, 13]. Therefore, most studies have ex- cluded by definition elderly patients from ERAS pathways. The reason for such exclusion was that adherence to protocols in elderly patients was assumed to be unfeasible due to Materials and methods Compliance with all interventions was combined and expressed as the percentage of patients who had correct inter- vention and documentation. Global compliance (GC) was de- fined as the rate of patients for whom compliance was achieved with all measures of the ERAS protocol. A retrospective analysis was performed from the GERM Group (Spanish for Grupo Español de Rehabilitacion Multimodal) prospective database by selecting patients ≥70 years, undergoing colon and rectal surgery following ERAS protocols in the last 2 years (2013–2014). Data was obtained from three university tertiary centers in Spain, Hospital Universitario Fundacion Jimenez-Diaz (Madrid), Hospital Clinico Universitario (Zaragoza), and Hospital General Universitario (Elche), of consecutive patients with elective colon and rectal resections excluding urgent and pal- liative surgeries. A multidisciplinary task force composed of surgeons, anesthesiologist, nurses, and physical therapists was convened in each center at the beginning of the study to im- plement and monitor compliance with interventions. A nurse coordinator was assigned to each center to routinely assess ERAS compliance obtained from the patient’s medical records and to report monthly the results to the study committee. In addition, a videoconference meeting was held every 3 months involving all three centers. The study was initiated after obtaining approval by the local institutional review board committee from each institution. The POSSUM score (Physiological and Operative Severity Score for the enumeration of Mortality and Morbidity) [18] was determined to calculate postoperative morbidity and mor- tality risks. Additional operative variables such as a stoma construction and the use of a laparoscopic surgical approach were also recorded. Short-term postoperative complications were graded using the Clavien-Dindo classification [15]. LOS and rates and causes of readmission during the first 30- day postoperative period were also documented. A further analysis was performed when LOS was prolonged to identify the causes of delayed hospital discharge. * Santiago Gonzalez-Ayora sgayora@fjd.es * Santiago Gonzalez-Ayora sgayora@fjd.es Int J Colorectal Dis (2016) 31:1625–1631 1626 the second postoperative day and early mobilization (walking from the bed to the sofa at 6–8 h after surgery), (9) intravenous fluid restriction and removal of urinary catheter (indicates stopping fluids and catheter removal by the first postoperative day), and (10) multimodal analgesia (epidural catheter for open surgery cases). physical impairments or associated comorbidities [14]. However, currently over 70 % of colorectal cancers are pri- marily diagnosed among patients over the age of 65 [15], and it seems reasonable that ERAS should be targeting elderly patients with associated comorbidities rather than young, healthy patients. A recent systematic review from the UK concludes that there currently exists a lack of evidence regard- ing ERAS in elderly patients and further studies are required to assess the implementation grade and possible benefits of ERAS protocols in this particular population [13]. By definition, we targeted hospital discharge date at fourth or fifth postoperative day for colon or rectal surgery, respec- tively. Patients could be discharged if they met the following criteria: good mobilization, adequate oral intake for liquids and solids, recovered gastrointestinal transit minimally for passing gas, normal urinary outputs, no wound problems, good pain control with oral medication, absence of fever in the last 48 h, C-reactive protein below 10 mg/L at discharge, and showing a decreasing trend in previous laboratory test. In addition, the patient and family needed to feel comfortable with the discharge and information given regarding possible complications and early detection. This led to the design of the present multicenter study to test the hypothesis of whether it is feasible to implement an ERAS program in elderly patients undergoing elective, colo- rectal surgery by assessing compliance with interventions and postoperative outcomes. Compliance outcomes Data was measured independently for each ERAS interven- tion as is presented in Table 2. By definition, we avoided mechanical preparation in colonic surgeries, and all patients received preoperative dietary recommendations and a carbohydrate-rich drink at 2–4 h before surgery. Early intake of clear liquids at 6 h after surgery and early mobilization were the most successfully carried out interventions in over 90 % of patients. On the other hand, the discontinuing early of intra- venous fluids and early removal of urinary catheter rates were 73 and 64 %, respectively. Statistical analysis Patient baseline characteristics at the time of surgery, age, gender, American Society of Anesthesiologists (ASA) score, major comorbidities, preoperative anticoagulation therapy were obtained from each patient’s electronic medical record. Compliance information was gathered from inpatient medical charts and recorded on a monthly basis in the database. Any measure during the process was considered as Bnon- compliant^ if documentation was incomplete. Descriptive sta- tistics are presented using t test with mean and standard devi- ation (SD) or median and range for continuous variables. Comparison of differences between group means was carried out using ANOVA for variables with normal distribution, and the Mann-Whitney test for continuous variables with non- parametric distribution. We used chi-squared analysis with Fisher’s exact test when any value observed in the contingen- cy table was less than 5 to compare proportion variables. We A bundle of 10 interventions were adopted at the same time in all three participating hospitals on the basis of our previ- ously published protocol [16, 17] including the following: (1) preoperative advice and evaluation of nutritional status, (2) intravenous iron supplementation in cases of preoperative anemia, (3) avoidance of full mechanical preparation for colon resections, (4) administration of carbohydrate-rich drinks 1 day prior and on the morning of surgery, (5) goal-directed intraoperative fluid therapy and body temperature control dur- ing surgery, (6) the use of intraoperative pneumatic legs com- pression, (7) avoidance of nasogastric tubes and drains when possible, (8) taking in oral fluids during the early postopera- tive period (means 6–8 h after surgery) and soft-food diet by 1627 Int J Colorectal Dis (2016) 31:1625–1631 performed a Kruskal-Wallis test to explore the impact of com- pliance within interventions on the length of stay. The level of statistical significance was taken as p < 0.05. All statistical calculations were performed using SPSS software® (version 22.0, SPSS Inc., Chicago, IL). The POSSUM scoring system was calculated for each pa- tient immediately after the surgery, with a median of 29.3 % [6.9–80.5] and 5.3 % [1.3–26.8] for expected postoperative morbidity and mortality, respectively. Results Throughout the study period a total of 188 patients were treat- ed among the three centers. Of them, 109 (58 %) were men, 79 (42 %) were women with a median age for the whole group of 79 [70–93] years old. Demographics, patient baseline charac- teristics, and surgical procedures are presented in Table 1. It is important to note that sigmoid resection and right and left colectomies were the most common surgery; 77 % (n = 145) performing a primary anastomosis. We also included rectal cancer surgeries (n = 43, 23 %); of them, 34 patients in whom low anterior resections were done with or without a temporary stoma. By summarizing these cases, 95 % of patients had a primary anastomosis with sphincter preservation. In addition, almost 45 % of surgeries (40 % colectomies; 58 % rectal surgeries) were performed via laparoscopic approach. Overall, there was a GC rate of 56 % of patients for whom compliance was achieved with all measured interventions. When performing laparoscopic surgery a higher, but non- significant GC percentage was achieved (59 % lap vs. 53.3 % open; p = 0.43). Patients who underwent rectal surgery had significantly lower GC rates when compared to colon surgery (41.9 % rectal vs. 60.0 % colon; p = 0.03). In addition, a construction of either a temporary or a definitive stoma, also decreased GC rates (36.0 % with stoma vs. 58.9 % without stoma; p = 0.03). Table 1 Patient baseline characteristics and surgical techniques Variable n = 188 Age (years)a 79 [70–93] Male 109 58 % Female 79 42 % POSSUM score (morbidity)a 29.3 % [6.9–80.5] POSSUM score (mortality)a 5.3 % [1.3–26.8] Comorbidities Diabetes 52 28 % Anticoagulant treatments 36 19 % Surgical technique Colon surgery 145 77.1 % Laparoscopic colon surgery 58 40.0 % Rectal surgery 43 22.9 % Laparoscopic rectal surgery 25 58.1 % Total laparoscopic surgery 83 44.1 % Surgical procedure Right colectomy 80 42.6 % Left colectomy 15 8.0 % Sigmoidectomy 50 26.6 % Low anterior resection 34 18.1 % Hartmann 7 3.7 % Abdominoperineal resections 2 1.1 % Stoma (temporary or definitive) 25 13.3 % a Median [range] Table 1 Patient baseline characteristics and surgical techniques Postoperative outcomes Postoperative outcomes, surgical complications, and length of stay are showed in Table 3. About 60 % of patients had an Table 2 Compliance rates with ERAS interventions Variable n (%) No drainagea 81 (43.0) Epidural anesthesiaa 116 (61.7) Early intake 173 (92.0) Early suspension of intravenous fluids 138 (73.4) Early mobilization 169 (89.9) Early urinary catheter removal 122 (64.9) Global compliance 105 (56.0) Variable Global compliance (%) X2 p value Laparoscopic surgery Open surgery 59.0 53.3 0.43 Colon surgery Rectal surgery 60.0 41.9 0.03 Without stomab With stomab 58.9 36.0 0.03 a ERAS interventions not included in the global compliance b Temporary or definitive stoma a Median [range] Int J Colorectal Dis (2016) 31:1625–1631 1628 non-significant effect of GC in terms of anastomotic leakage was present (7.6 % with GC vs. 8.4 % without GC; p = 0.83). uneventful postoperative course without complications (66.7 % when GC achieved vs. 56.6 % without GC), 25 % had minor complications, prolonged ileus being the most common complication. Important to note, we did not observe frequent medical complications in elderly patients such as postoperative pneumonia or venous thromboembolism. Major complications occurred in 13 % of cases, including 15 patients who had a clinical anastomotic leakage (8 %). The need for a reoperation was 8.5 % (16 patients) mainly due to anastomotic leakage. Mortality following complica- tions occurred in three patients (1.6 %). As expected, patients who had rectal surgeries suffered worse complications com- pared to patients with colon surgeries and with a higher risk of anastomotic leakage. uneventful postoperative course without complications (66.7 % when GC achieved vs. 56.6 % without GC), 25 % had minor complications, prolonged ileus being the most common complication. Important to note, we did not observe frequent medical complications in elderly patients such as postoperative pneumonia or venous thromboembolism. Major complications occurred in 13 % of cases, including 15 patients who had a clinical anastomotic leakage (8 %). The need for a reoperation was 8.5 % (16 patients) mainly due to anastomotic leakage. Mortality following complica- tions occurred in three patients (1.6 %). As expected, patients who had rectal surgeries suffered worse complications com- pared to patients with colon surgeries and with a higher risk of anastomotic leakage. Median LOS was 6 [3–51] days for the entire study population including the readmission days when occurred. Postoperative outcomes A significant im- pact of GC on LOS was observed with a median reduction of 1 day in LOS including readmissions (6 [3–30] days with GC vs. 7 [4–51] days without GC; p = 0.03).Figure 1 shows the influence of percentage of GC in reduction of median LOS. A significant reduction of 1.5 days in LOS was achieved when we were able to reach a 50 % rate in GC (7.5 [5–51] days < 50 %GC vs. 6 days [3–30] days > 50 %;p < 0.04]Figure 2 shows further analysis of LOS based on the postoperative day, showing that 41 % of the study population were discharged at ERAS or ERAS + 1 day periods. The main reasons for a delayed discharge are shown in Fig. 2. indicating social issues to be the most observed reasons for delay in discharge compared to postoperative complications. Readmission to the hospital occurred in 12 patients (6.4 %). The most common cause of readmission was deep and organ/ space (intra-abdominal) infections requiring CT-guided percuta- neous drainage and/or antibiotic coverage. There were two cases of late anastomotic leakage with pelvic abscesses in patients who underwent low anterior resections When investigating the impact of GC on postoperative complications, we observed the following data as shown in Table 3: (1) a clinical, but not statistically significant 10 % reduction on minor Clavien-Dindo I/II complications (30 % with GC vs. 20 % without GC, p = 0.32). While we did not observed influences of achieving GC on major Clavien-Dindo III/IV complications (13.4 % with GC vs. 13.2 % without, p = 0.34), (2), a lower percentage of postoperative ileus (21 % with GC vs. 26.5 % without GC; p = 0.37), and (3) a Discussion Table 3 Postoperative outcomes Postoperative complications n (%) X2 p value No complications 117 (62.2) Dindo-Clavien grades I–II 46 (24.5) with GC 30 without GC 20 0.32 Dindo-Clavien grades III–V 25 (13.3) with GC 13.4 without GC 13.2 0.34 Postoperative ileus 44 (23.4) with GC 21.0 without GC 26.5 0.37 Anastomotic leakage 15 (8.0) with GC 7.6 without GC 8.4 0.83 Mortality 3 (1.6) Reoperations Anastomotic leakage Bowel ischemia Hemoperitoneum Internal hernia Urinary tract injury 16 (8.5) 10 (5.3) 2 (1.0) 1 (0.5) 1 (0.5) 1 (0.5) Hospital length of staya 6.0 [3–51] with GC 6 [3–30] without GC 7 [4–51] 0.03 Readmissions Abdominal abscess Wound infection Late anastomotic leakage Dehydration 12 (6.4) 3 (1.6) 3 (1.6) 2 (1.0) 3(1.0) a Median [range] Table 3 Postoperative outcomes Our data shows reliable outcomes from a multicenter, obser- vational, non-randomized study group in the implementation of a standardized ERAS protocol in elective colorectal surgery in elderly patients. There is a lack of information on the exact impact of ERAS interventions in elderly patients, although the current evidenced-based data has been recently reviewed in a system- atic review from the UK [13]. This review reported to date two clinical trials comparing ERAS with non-ERAS, focused on elderly patients showing in favor of ERAS, a shorter length of stay and a significant decrease in minor complications [19, 20]. However, one of these studies had a low number of pa- tients, did not show data about rates of reoperation or read- mission, and did not report compliance with interventions. Due to lacking such data, our study proved that a sustained effort from a dedicated multidisciplinary team could achieve a high level of compliance rates with most ERAS interventions in both colon and rectal surgeries focused in elderly patients. When analyzing compliance with ERAS bundles, we ob- tained the goal of 90 % compliance within two interventions: early intake of clear liquids and early mobilization when planned. Both were difficult to be implemented and required an extra effort from the caregivers, as most elderly patients were reluctant to get out of bed or drink liquids 6–8 h after surgery. In our experience, management of proper pain control within over 60 % of patients with epidural morphine-sparing analgesia can help to accomplish early mobility from the bed 1629 Int J Colorectal Dis (2016) 31:1625–1631 * * * p<0.05 < 50% of GC LOS: 7.5 days [5-51] vs. Discussion When analyzing the impact of GC in postoperative complications, there was a clinical, but not significant reduction of 10 % in minor com- plications and about 5–6 % in postoperative ileus, while may- or postoperative complications such as anastomotic leakage remained unchanged. Our aim was to establish and detect an impact of GC in decreasing complications; however, a small sample size may have underpowered the effect of GC in com- plications in our study. Readmission to the hospital after discharge was ob- served in 6.4 % of patients, mainly due to abdominal abscess after pelvic surgeries. We reported two cases of late anastomotic leakage in rectal cancer surgeries in pa- tients who were readmitted to the hospital needing percu- taneous drainage. In contrast, there were no cases of de- layed leakage in colon resections. We believe that, based on the study population of elderly patients, these LOS and readmissions rates are considerably good and support the idea that ERAS is a feasible and secure option for this particular population. For predicting patients at risk of developing complications, we assessed for each patient their POSSUM score. We be- lieved that the POSSUM score might be more useful than the ASA score to predict postoperative outcomes and that it is adequate to assess patient’s baseline performance status; however, it is not a valid tool to identify patients at risk of failure in the ERAS programs. The key point would be to select in the preoperative evaluation who will fail in achieving ERAS, to either design a personalized program for those pa- tients, or have the opportunity to improve their conditions in the prehabilitation period. In this sense, a better score taking into account Bfrailty^ rather than Belderly^ using the modified frailty index has demonstrated to correlate better with compli- cations, longer lengths of stay, and readmissions and has re- cently been validated in elderly patients undergoing colorectal surgery under ERAS protocols [22]. Therefore, we would recommended before implementing ERAS in elderly patients to use a prospective score to identify patients at risk for not achieving the protocol so resources and postoperative sup- ports would be better allocated. Our study has some limitations that deserve to be men- tioned. First, although this was a multicenter study, it was not being conducted, as a randomized clinical trial, and the number of cases was small in order to established robust conclusions. Discussion LOS is often used as a surrogate marker of recovery, and it should not be offset by a higher rate of hospital readmission. GC with statistical differences were rectal surgeries, the crea- tion of a stoma and open surgery cases. Therefore, a great effort should be made to increase our plan of care in patients with these characteristics. In our data, the median LOS was 6 days for the entire study population with minimal differences between hospitals. When analyzing LOS by subgroups, 41 % of patients were discharge at the estimated day in ERAS protocols (in the fourth postop- erative day for colon surgery or fifth postoperative day for rectal surgery) or ERAS + 1 day. Moreover, when analyzing the reasons for a delayed discharge, a needed of social support and non-postoperative complications were the most common causes for patients to be discharged from the hospital before the 10th postoperative day. We believe this to be because two of the hospitals from the study are reference hospitals covering suburban and rural areas. These patients may live a greater distance from the hospital, making physicians reluctant to dis- charge them earlier. Important to note, our data showed a positive effect of GC in LOS when we were able to achieve, at least, 50 % of compliance with the interventions. Criticism of ERAS protocols will argue that a high read- mission rate especially in elderly patients will invalidate any positive result. Regarding 30-day postoperative outcomes, our data showed that 62 % of patients had no complications, 25 % had minor complications, and 13 % suffered major Clavien- Dindo’s complications. Postoperative ileus was the most com- mon observed complication in almost 25 % of patients who required a nasogastric tube and prolonged total parenteral nu- trition. We did not observe respiratory complications such as pneumonia or pulmonary edema or cases of deep vein throm- bosis. Reoperation was needed in 8.5 % of patients and clin- ical anastomotic leakage occurred in 8 % for colon surgeries and 11.6 % in rectal surgeries. An impact of GC on decreasing postoperative complica- tions has been earlier reported in 2011 by a multicenter study from the European ERAS Study Group especially when GC could be achieved by over 70 % [21]. Discussion >50% of GC LOS: 6 days [3-30]; p=0.004 Fig. 1 Influence of Compliance with ERAS measures in LOS (Kruskal-Wallis test) ( ) Fig. 1 Influence of Compliance with ERAS measures in LOS (Kruskal-Wallis test) Fig. 1 Influence of Compliance with ERAS measures in LOS (Kruskal-Wallis test) * p<0.05 < 50% of GC LOS: 7.5 days [5-51] vs. >50% of GC LOS: 6 days [3-30]; p=0.004 to the sofa without adding secondary effects. Moreover, al- most half of the participants where operated via laparoscopic approach, adding advantages in achieving these compliance rates. From the rest of ERAS interventions, early stopping of intravenous fluids and early urinary catheter removal showed the lowest compliance rates (73 and 65 %, respectively). We believe the reason for this delay was due to short urinary outputs in elderly patients during the first postoperative day leading to a delayed removal of the urinary catheter. Instead of looking at each specific intervention, we developed the vari- able global compliance, defined as the rate of patients for whom compliance was achieved with all the measurements of the ERAS protocol. Overall, there was a GC rate of 56 % in the study population. Identified barriers to achieve a higher 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% ED* + 2 days Discharge before 10 days PLOS* Major complicaons Minor complicaons Social causes * Esmated discharge in ERAS protocol. Discharge before 10 days excluding ED / ED + 1 / ED + 2. * Prolonged length of stay (discharge ≥ 10 days). Fig. 2 Reasons for delaying discharge beyond ERAS estimated date. Estimated discharge in ERAS protocol 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% ED + 2 days Discharge before 10 days PLOS* Major complicaons Minor complicaons Social causes * Esmated discharge in ERAS protocol. Discharge before 10 days excluding ED / ED + 1 / ED + 2. * Prolonged length of stay (discharge ≥ 10 days). 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% ED* + 2 days Discharge before 10 days PLOS* Major complicaons Minor complicaons Social causes * Esmated discharge in ERAS protocol. * Esmated discharge in ERAS protocol. Int J Colorectal Dis (2016) 31:1625–1631 1630 reported a mean LOS of 3.9 days [24]. Discussion Secondly, we did not compare our results in elderly patients to a control group of patients <70 years old with the same ERAS protocol or under the traditional treat- ment, due to a lack of information in our previous database prior to the start of the study. References 17. Esteban F, Cerdan FJ, Garcia-Alonso M, Sanz-Lopez R, Arroyo A, Ramirez JM, Moreno C, Morales R, Navarro A, Fuentes M (2013) A multicentre comparison of a fast track or conventional postoper- ative protocol following laparoscopic or open elective surgery for colorectal cancer surgery. Color Dis 16:134–140 1. Heald RJ, Husband EM, Ryall RD (1982) The mesorectum in rectal cancer surgery—the clue to pelvic recurrence? Br J Surg 69:613–616 2. Biondi A, Grosso G, Mistretta A, Marventano S, Toscano C, Drago F, Gangi S, Basile F (2013) Laparoscopic vs. open approach for colorectal cancer: evolution over time of minimal invasive surgery. BMC Surg 13(Suppl 2):S12 18. Copeland GP, Jones D, Walters M (1991) POSSUM: a scoring system for surgical audit. Br J Surg 78:355–360 19. Wang Q, Suo J, Jiang J, Wang C, Zhao YQ, Cao X (2012) Effectiveness of fast-track rehabilitation vs conventional care in laparoscopic colorectal resection for elderly patients: a randomized trial. Color Dis 14:1009–1013 3. Morneau M, Boulanger J, Charlebois P, Latulippe JF, Lougnarath R, Thibault C, Gervais N (2013) Laparoscopic versus open surgery for the treatment of colorectal cancer: a literature review and rec- ommendations from the Comite de l'evolution des pratiques en oncologie. Can J Surg J Canadien de Chirurgie 56:297–310 20. Jia Y, Jin G, Guo S, Gu B, Jin Z, Gao X, Li Z (2014) Fast-track surgery decreases the incidence of postoperative delirium and other complications in elderly patients with colorectal carcinoma. Langenbeck's Arch Surg / Deutsche Gesellschaft fur Chirurgie 399:77–84 4. Kehlet H, Wilmore DW (2002) Multimodal strategies to improve surgical outcome. Am J Surg 183:630–641 g g 5. Kehlet H, Wilmore DW (2005) Fast-track surgery. Br J Surg 92:3–4 6. Wind J, Polle SW, Fung Kon Jin PH, Dejong CH, von Meyenfeldt MF, Ubbink DT, Gouma DJ, Bemelman WA (2006) Systematic review of enhanced recovery programmes in colonic surgery. Br J Surg 93:800–809 21. Gustafsson UO, Hausel J, Thorell A, et al. (2011) Adherence to the enhanced recovery after surgery protocol and outcomes after colo- rectal cancer surgery. Arch Surg 146(5):571–577 22. Keller DS, Bankwitz B, Nobel T, Delaney CP (2014) Using frailty to predict who will fail early discharge after laparoscopic colorectal surgery with an established recovery pathway. Dis Colon rectum. Mar 57(3):337–342. 7. Conflict of interest The authors declare that they have no conflict of interest. 12. Polle SW, Wind J, Fuhring JW, Hofland J, Gouma DJ, Bemelman WA (2007) Implementation of a fast-track perioperative care pro- gram: what are the difficulties? Dig Surg 24:441–449 13. Bagnall NM, Malietzis G, Kennedy RH, Athanasiou T, Faiz O, Darzi A (2014) A systematic review of enhanced recovery care after colorectal surgery in elderly patients. Color Dis 16:947–956 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 appro- priate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 14. Pawa N, Cathcart PL, Arulampalam THA, Tutton MG, Motson RW (2012) Enhanced recovery program following colorectal resection in eldery patient. World J Surg 36:415–423 15. Dindo D, Demartines N, Clavien PA (2004) Classification of sur- gical complications: a new proposal with evaluation in a cohort of 6336 patients and results of a survey. Ann Surg 240:205–213 16. Ramirez JM, Blasco JA, Roig JV, Maeso-Martinez S, Casal JE, Esteban F, Lic DC (2011) Enhanced recovery in colorectal surgery: a multicentre study. BMC Surg 11:9 Author’s contributions Santiago Gonzalez-Ayora and Carlos Pastor contributed equally to analyze the data and write the manuscript. Author’s contributions Santiago Gonzalez-Ayora and Carlos Pastor contributed equally to analyze the data and write the manuscript. Acknowledgments The authors thank Sara Rosenstone Calvo and Patricia Tejedor M.D. for editing this manuscript. Acknowledgments The authors thank Sara Rosenstone Calvo and Patricia Tejedor M.D. for editing this manuscript. 10. Liang LV, Sao Y-f, Zhou Y-b (2012) The enhanced recovery after surgery (ERAS) pathway for patients undergoing colorectal sur- gery: an update of meta-analysis of randomized controlled trials. Int J Color Dis 27:1549–1554 Conclusions Based on our data from the present multicenter study, ERAS should be implemented without reservations in elderly pa- tients undergoing elective colorectal surgeries, expecting the same goals and benefits as found in other age groups. Barriers in achieving a high compliance with ERAS interventions in elderly patients are common and will require a great effort in the patient education, an intensive plan of pre and postopera- tive care, and sometimes a change in the surgeons’ management. Contemporary postoperative admission stays in ERAS pro- tocols range from 3 to 5 days [23] in comparison to traditional practice of up to 5 to 9 days. Focused on surgery in elderly patients, a prospective study of 87 patients >70 years old, Int J Colorectal Dis (2016) 31:1625–1631 1631 decrease long-term survival but not the time to recurrence. Color Dis Feb. 17(2):141–149. Compliance with ethical standards 11. Lohsiriwat V (2014) Impact of an enhanced recovery program on colorectal cancer surgery. Asian Pac J Cancer Prev 15:3825–3828 Conflict of interest The authors declare that they have no conflict of interest. Conflict of interest The authors declare that they have no conflict of interest. References Adamina M, Kehlet H, Tomlinson GA, Senagore AJ, Delaney CP (2011) Enhanced recovery pathways optimize health outcomes and resource utilization: a meta-analysis of randomized controlled trials in colorectal surgery. Surgery 149:830–840 23. DiFronzo LA, Yamin N, Patel K (2003) Benefits of early feeding and early hospital discharge in elderly patients undergoing open colon resection. J Am Coll Surg 197:747 8. Spanjersberg WR, Reurings J, Keus F, van Laarhoven CJHM (2011) Fast track surgery versus conventional recovery strategies for colorectal surgery (review). The Cochrane Library Issue 2 24. DiFronzo LA, Yamin N, Patel K (2003) Benefits of early feeding and early hospital discharge in elderly patients undergoing open colon resection. J Am Coll Surg 197:747 9. Odermatt M, Miskovic D, Flashman K, Khan J, Senapati A, O'Leary D, Thompson M, Parvaiz A (2015) Major postoperative complications following elective resection for colorectal cancer
W2923223284.txt	https://europepmc.org/articles/pmc6597732?pdf=render	en		Future projection of cancer patients with cardiovascular disease in Japan by the year 2039: a pilot study	International journal of clinical oncology	2,019	cc-by	6,396	International Journal of Clinical Oncology (2019) 24:983–994 https://doi.org/10.1007/s10147-019-01426-w ORIGINAL ARTICLE Future projection of cancer patients with cardiovascular disease in Japan by the year 2039: a pilot study Yuji Okura1 · Tsugumi Takayama2 · Kazuyuki Ozaki2 · Hiroshi Tanaka3 · Akira Kikuchi4 · Toshihiro Saito5 · Toshiki Tanigawa5 · Yasumasa Takii6 · Hiroshi Seki7 · Tatsuya Takenouchi8 · Takaaki Chou9 · Nobuaki Sato10 · Naohito Tanabe11 · Tohru Minamino2 Received: 14 September 2018 / Accepted: 1 March 2019 / Published online: 22 March 2019 © The Author(s) 2019 Abstract Background The number of cancer patients in Japan is estimated to rise to 3.5 million by 2025. The disease burden may be further complicated by comorbidities caused by cardiovascular disease (CVD). Predicting the number of cancer patients with CVD can help anticipate future resource needs. Methods We used statistics derived from the Niigata Cancer Center CVD Study (2015) as well as population estimates from the National Cancer Center’s Cancer Registry and Statistics survey of 2017 for convenience. We simply multiplied the projected number of cancer patients through the year 2039 by the CVD prevalence in 2015, with patients classified by sex, age, and cancer type to estimate the number of cancer patients with CVD. Results The total number of Japanese cancer patients with CVD was 253,000 in 2015 and is predicted to increase rapidly by 30,000 in 2020 and peak at 313,000 in 2030–2034. Men will dominate the CVD population at 2.5-fold the number of women. The growth rate of the population with both cancer and CVD will be greater than that of the cancer-only population (1.23 vs 1.18, P < 0.001), and will comprise notably high proportions of patients with prostatic, breast, and uterine cancers (1.80, 1.57, and 1.66, P < 0.001, respectively). Conclusion Future cancer patients will be older and more likely to have CVD. Although men will continue to dominate this population, the increase in the number of women will be pronounced. Cancer care providers should be trained to recognize CVD and provide any necessary interventions concurrently with cancer therapy. Keywords Epidemiology · Cancer · Cardiovascular disease · Comorbidity · Prediction models Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10147-019-01426-w) contains supplementary material, which is available to authorized users. * Yuji Okura okuray@niigata‑cc.jp 1 2 Department of Onco‑cardiology, Niigata Cancer Center Hospital, Chu‑o‑ku Kawagishicho 2‑15‑3, Niigata 951‑8560, Japan Department of Cardiovascular Biology and Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan 6 Department of Gastroenterological Surgery, Niigata Cancer Center Hospital, Niigata, Japan 7 Department of Diagnostic Radiology, Niigata Cancer Center Hospital, Niigata, Japan 8 Department of Dermatology, Niigata Cancer Center Hospital, Niigata, Japan 9 Department of Hematology, Niigata Cancer Center Hospital, Niigata, Japan 3 Department of Respiratory Medicine, Niigata Cancer Center Hospital, Niigata, Japan 10 Department of Breast Oncology, Niigata Cancer Center Hospital, Niigata, Japan 4 Department of Gynecology, Niigata Cancer Center Hospital, Niigata, Japan 11 Department of Health and Nutrition, Faculty of Human Life Studies, University of Niigata Prefecture, Niigata, Japan 5 Department of Urology, Niigata Cancer Center Hospital, Niigata, Japan 13 Vol.:(0123456789) 984 International Journal of Clinical Oncology (2019) 24:983–994 Introduction Methods Cancer and cardiovascular disease (CVD) are major public health problems with rising incidence rates in developed countries [1–4]. In 2015, approximately 3.1 million individuals in Japan had cancer, and more than 862,000 new cases are diagnosed annually [3]. Separately, the number of outpatients with left ventricular dysfunction (LVD) was 979,000 in 2005 and is predicted to increase gradually as the population ages, reaching 1.3 million by 2030 [4]. CVD is endemic among cancer patients; hypertension, atrial fibrillation (AF), ischemic heart disease (IHD), venous thromboembolism (VTE), LVD, and heart failure (HF) are common comorbidities [5, 6]. This is attributable to common risk factors shared by cancer and CVD (e.g., aging, smoking, diabetes, obesity, and physical inactivity), neoplastic effects on the cardiovascular system, chemotherapy- and radiotherapyinduced cardiovascular toxicities, and longer life expectancies [1, 3, 6–9]. CVD-related comorbidities are a persistent burden on cancer patients [5–7]. CVD causes acute HF, myocardial infarction, pulmonary thromboembolism, stroke, cardiogenic shock, syncope, and arrhythmia; these can be lethal or lead to serious disabilities. CVDs often relapse, and some cause major adverse cardiovascular events (MACEs); they also often interrupt the patient’s cancer treatment. Some anticancer drugs are contraindicated to avoid CVD and MACE, resulting in insufficient treatment and a worse prognosis. For example, anthracycline and trastuzumab are withheld for LVD patients, while bevacizumab is withheld for VTE patients. Curative surgery may be abandoned in favor of palliative surgery or radiotherapy. In our registry, the 5-year survival rate was 64.0% for all cancer patients and 44.2% for cancer patients with CVD [6]. Comorbidities with AF, VTE, and N-terminal prohormone of brain natriuretic peptide (NT-proBNP) elevation were significantly associated with mortality [6]. The number of cancer patients in Japan is estimated to increase to 3.5 million by 2025 [3]. The future disease burden may be further complicated by comorbidities associated with CVD and may vary among cancer types. Predicting the CVD epidemic can help to anticipate future resource needs among cancer-treating institutions. However, to our knowledge, no predictive studies have been performed on this issue in Japan. Therefore, we conducted a retrospective study at our cancer center hospital to predict the impact of CVD on cancer patients over the next 20 years. Study setting and design To predict the future number of cancer patients with CVD, we utilized the projected number of cancer patients by 2039 as determined by national cancer statistics (Supplementary Table 1) [3]. The CVD prevalence in 2015 was determined by examining our institutional cancer registry, in which we identified all cancer patients with CVD over a 10-year period (2005–2014), as previously described [6]. We also estimated CVD prevalence as correlated with age, sex, and cancer type in 2015 (Supplementary Table 2), and then extrapolated these correlations to project the number of cancer patients in each age and sex group up to 2039 [3] and to estimate the number of cancer patients with CVD. Institutional cancer database Our hospital maintains a cancer registry that is regularly updated with the clinical information and vital statuses of patients. The dates of initial diagnosis, clinical and pathological diagnosis, and death were obtained from the registry [6, 9]. Selection of patients with CVD Systemic screening at our hospital is performed before cancer therapy, as previously described [6, 9]. All patients underwent a physical examination, 95% submitted to electrocardiograms, and 53% underwent chest radiographies. When necessary, cancer patients were referred to cardiologists; 23% and 7% of the patients underwent transthoracic echocardiography and vascular echo, respectively. Nearly all of the patients (98%) underwent computed tomography (CT) to detect tumors in the chest, abdomen, or pelvis. NTproBNP levels were measured for screening or diagnosis of HF beginning in 2008. These data were double-checked and stored in our database. Only the first image or record was analyzed for patients with multiple imaging studies or laboratory tests that satisfied our inclusion criteria. Moreover, the maximum values of NT-proBNP obtained for each patient were selected for analysis. Definition and diagnosis of CVD We focused on LVD, AF, IHD, aortic stenosis (AS), VTE, and significant elevation of NT-proBNP because these conditions constitute a burden on cancer patients seeing as they 13 International Journal of Clinical Oncology (2019) 24:983–994 require sustained surveillance or medication. Although hypertension was the most common CVD in both cancer and non-cancer patients, we did not focus on it owing to its wide prevalence and relatively successful treatability. The definitions of CVDs used were the same as those described previously, as follows [6]: LVD was divided into systolic dysfunction (LVSD) and diastolic dysfunction (LVDD) exclusively, as determined by echocardiography. LVSD was diagnosed based on a left ventricular ejection fraction (LVEF) ≤ 50%, while LVDD was diagnosed based on an ejection fraction > 50% and early transmitral flow velocity/early diastolic velocity of the mitral annulus > 8 plus at least 2 of the following criteria: (1) left atrial enlargement, (2) left ventricular hypertrophy, and (3) AF [9]. AF was diagnosed using 12-lead electrocardiography. IHD was identified if the patient had at least 2 of the following: (1) documented history of IHD (myocardial infarction, angina, or prior coronary revascularization); (2) medication for IHD, and (3) presence of IHD following any cardiac tests or imaging. AS was classified as moderate or severe depending on the echocardiography findings [9]. VTE was diagnosed if proximal deep venous thrombosis (DVT) or pulmonary thromboembolism were present. VTE was diagnosed with contrast-enhanced CT by radiologists. DVT of the legs was also diagnosed with vascular echo. We included all pulmonary thromboemboli and central types of DVT (superior vena cava, inferior vena cava, subclavian, iliac, femoral, and popliteal DVT); however, we excluded DVT of the lower legs. NT-proBNP > 900 pg/mL was considered a significant elevation based on the Japanese Heart Failure Society statement and international standards [10, 11]. CVD was defined as any of LVSD, LVDD, AF, IHD, AS, VTE, or elevation of NT-proBNP > 900 pg/mL that were documented anytime during the clinical course. 985 Projected number of cancer patients with CVD per each cancer type We applied the CVD prevalence reported in 2015 [6] to the projected number of cancer patients in each age and sex group until the year 2039 [3] to provide a prospective estimate of the number of cancer patients with CVD. The projected number of cancer patients [3] was calculated using the formula reported by Pisani et al. [12] with incidence rates and survival probabilities in Japan [13]. To predict the total number of cancer patients with each type of CVD, we used a twostep process as follows. Using AF as an example, we first determined the subtotals of the cancer patients with AF for each cancer type. Second, we summed the subtotals of each aforementioned cancer type and presented the sums as the predicted total number of cancer patients with AF. These steps were repeated for each type of CVD within each time period. Statistical analysis The proportions of cancer with each CVD were compared between 2015–2019 and 2035–2039 using χ2 test. The change in the number of patients with each cancer type from 2015–2019 to 2035–2039 (growth rate) was calculated using the following equation: Number of cancer patients in 2035−2039∕ number of cancer patients in 2015−2019. The odds ratio of comorbidity of CVD in 2035–2039 in comparison with 2015–2019 was calculated for each cancer. Data were analyzed with IBM SPSS statistics version 25.0 (Armonk, NY, USA). Ethical considerations Prevalence of CVD according to cancer type We determined the number of cancer patients who had CVD between 2005 and 2014 and who survived until January 2015. The proportion of CVD patients among cancer survivors in 2015 was considered the prevalence of CVD in the present study (Supplementary Table 2) [6]. If any patient was diagnosed with any other cancers 2 or more times during the recruitment period and survived until January 2015, CVD was counted for each cancer type to determine the cancer type-specific CVD prevalence. Cancer types were categorized in accordance with International Classification of Diseases, 10th revision (Supplementary Table 1–3). There were too few patients with male breast cancer (n = 4) and brain cancer (n = 5) to evaluate CVD prevalence. Other cancers and unspecified primary sites were not included in the study. The study protocol was reviewed and approved by the Niigata Cancer Center ethics committee. The study was approved as minimal-risk research and informed consent was waived in accordance with the ethical guidelines for epidemiology research in Japan. Results Prevalence of CVD among cancer patients in 2015 Table 1 shows the prevalence of CVD across 6 age groups. LVSD, LVD, and elevation of NT-proBNP were frequently observed in patients with childhood and adolescent cancer. Generally, the rate of CVD increased progressively with age, reaching 13.1% in male patients and 7.9% in female patients. 13 986 International Journal of Clinical Oncology (2019) 24:983–994 However, VTE rates were largely unchanged among age groups. AS increased progressively with age and occurred more frequently in female than in male patients. The prevalence data of each type of CVD according to the type of cancer are shown in Supplementary Table 2. Projection of cancer patients with CVD by 2039 Japanese cancer patients totaled 3.1 million in 2015; their numbers were expected to increase rapidly by 300,000 by 2020 and peak at 3.5 million in 2025–2034 (Fig. 1 and Supplementary Table 1). The total number of Japanese cancer patients with CVD was 253,000 in 2015 and is expected to rise by 30,000 in 2020, peaking at 313,000 in 2030–2034 (Fig. 1 and Supplementary Table 3). Male patients will dominate the CVD population as a whole, at 2.5-fold greater than the female population. Male CVD will peak by the early 2030s; however, female CVD will continue to increase thereafter. AF was the most common CVD in cancer patients in 2015 (111,000 people) and its incidence is expected to peak at 137,000 in 2030. AF in men will be 4 times more prevalent than in women. The majority of cancer patients with AF were over 75 years (52% and 54% of male and female Table 1 Prevalence of CVD in all cancer (C00-96) in age groups Male Age groups LVSD LVDD LVD AF IHD AS VTE NT-proBNP CVD Female Age groups LVSD LVDD LVD AF IHD AS VTE NT-proBNP CVD Fig. 1 Projected number of cancer patients between 2015 and 2039.▸ Bar graphs show the number of cancer patients in total and with each CVD stratified by 5-year intervals. The numbers of cancer patients are the sum of “all (C00–C96)” lines listed in Supplementary Table 1. The numbers of cancer patients with CVD are listed in the “total” categories of Supplementary Table 3. AF, atrial fibrillation; AS, aortic stenosis; CVD, cardiovascular disease; IHD, ischemic heart disease; LVD, left ventricular dysfunction; LVDD, left ventricular diastolic dysfunction; LVSD, left ventricular systolic dysfunction; NT-proBNP, N-terminal pro-brain natriuretic peptide; VTE, venous thromboembolism patients, respectively). LVD was the second most common CVD in patients with cancer, numbering 87,000 in 2015; this number is expected to peak at 106,000 in 2030. Most cancer patients with LVD were over 75 years of age (51% and 53% of male and female patients, respectively). IHD was the third most common comorbidity among cancer patients with CVD; a strong male predominance (6.5-fold that of female patients) will be observed in the future. There were 61,000 cancer patients with IHD in 2015; this number is expected to peak at 72,000 in 2030. 0–14 3.49 0.00 3.49 1.16 0.00 0.00 1.16 11.63 12.79 15–44 0.51 0.25 0.76 0.25 0.76 0.00 1.26 0.25 2.78 45–54 0.84 0.56 1.40 1.26 1.54 0.00 1.26 0.56 5.04 55–64 0.77 1.54 2.31 2.72 2.31 0.08 0.65 0.85 7.14 65–74 1.53 1.91 3.44 4.93 3.22 0.48 1.02 1.24 10.44 752.20 2.41 4.61 6.71 3.46 1.20 1.31 3.20 13.10 All 1.31 1.58 2.89 3.98 2.46 0.39 0.98 1.36 8.73 0–14 5.77 0.00 5.77 3.85 0.00 0.00 1.92 7.69 19.23 15–44 0.13 0.20 0.34 0.00 0.07 0.00 0.54 0.07 0.87 45–54 0.19 0.19 0.38 0.13 0.06 0.00 0.38 0.25 0.89 55–64 0.27 0.64 0.91 0.37 0.46 0.09 1.37 0.32 2.97 65–74 0.40 1.45 1.85 2.40 0.60 0.75 1.30 1.05 6.00 750.81 1.94 2.75 2.91 0.97 1.78 1.13 2.10 7.92 All 0.39 0.79 1.18 1.05 0.40 0.44 0.95 0.72 3.53 C00-96 indicates cancer types coded by ICD 10. CVD prevalence in all cancers (from 00 to 96) are derived from the Niigata Cancer Center study (2015). Values in each CVD line are percentages AF atrial fibrillation, AS aortic stenosis, CVD cardiovascular disease, IHD ischemic heart disease, LVD left ventricular dysfunction, LVDD left ventricular diastolic dysfunction, LVSD left ventricular systolic dysfunction, NT-proBNP N-terminal pro-brain natriuretic peptide, VTE venous thromboembolism 13 International Journal of Clinical Oncology (2019) 24:983–994 987 13 988 International Journal of Clinical Oncology (2019) 24:983–994 Fig. 1 (continued) Proportion of cancer types in male CVD patients in 2015 and 2039 The top 5 male cancers in 2015 were prostate, stomach, colorectal, lung, and kidney/ureter (Supplementary Table 1, Fig. 2 ‘Cancer’ panel). The number of prostate cancer patients will increase 1.6-fold overall (Table 2) and 1.4-fold as a proportion of male cancers by 2035 (Fig. 2); the rank will change to 2nd or 3rd as colorectal cancer rates will increase while stomach cancer rates will decline by 2035. In general, a high CVD prevalence in elderly patients with various cancer types (Supplementary Table 2) increased the prevalence rank of each cancer (e.g., colorectal cancer in LVD, IHD, VTE, high NT-proBNP, or CVD, and urinary bladder cancer in LVD), while an average CVD prevalence in frequent cancers retained the latter’s higher proportions (e.g., prostate cancer with SD, AF, or CVD) in 2015. As cancer patients age, those with prostate cancer plus CVD will increase 1.5-fold in proportion by 2035 (Fig. 2) and 1.8-fold in number (Table 2; Fig. 3). Proportion of cancer type in female CVD patients in 2015 and 2039 The top 5 cancers in 2015 were breast, colorectal, stomach, uterus, and lung (Supplementary Table 1, Fig. 2 ‘Cancer’ panel). In contrast to men, the proportion of women with cancer is projected to remain relatively unchanged. In general, a high CVD prevalence in elderly patients with various cancer types increased their rank proportions (e.g., colorectal cancer with SD, IHD, or VTE, as well as uterine cancer with LVDD, LVD, VTE, or CVD). Moreover, an average CVD prevalence in frequent cancers maintained their high rank proportions (e.g., breast cancer with LVDD, LVD, AF, AS, high NT-proBNP, or CVD) in 2015. Patients with VTE and high NT-proBNP had notably higher rates of gynecologic cancers and hematological 13 Fig. 2 Proportions of the top 7 cancer types in 2015–2019 and▸ 2035–2039 in cancer patients with each type of CVD. The numbers of cancer patients are the sum of each categories from ‘Oral cavity & pharynx’ (C00–C14) to ‘Leukemia’ (C91–C95) listed in Supplementary Table 3. The numbers of cancer patients with CVD are listed in the “total” categories of Supplementary Table 3. According to Japanese national cancer statistics, there were 1.7 million male cancer patients and 1.4 million female cancer patients in 2015; the numbers are expected to be 1.9 million and 1.7 million in 2025, respectively (Supplementary Table 1). To compare the proportion of cancer types, we summed the number of patients with each cancer and considered the total to be the denominator. Accordingly, the number of cancer patients listed here was larger than those of Japanese national cancer statistics. AF atrial fibrillation, AS aortic stenosis, CVD cardiovascular disease, IHD ischemic heart disease, LVD left ventricular dysfunction, LVDD left ventricular diastolic dysfunction, LVSD left ventricular systolic dysfunction, NT-proBNP N-terminal pro-brain natriuretic peptide, VTE venous thromboembolism malignancies, respectively. As cancer patients age, breast cancer patients with CVD will increase 1.2-fold in proportion (Fig. 2) and 1.6-fold in number by 2035 (Table 2; Fig. 3). Growth rates of top 5 cancers with and without CVD The growth rates of the top 5 cancers with and without CVD were compared between 2015–2019 and 2035–2039 (Table 2). The growth rate of cancer with CVD is predicted to be larger than that of cancer without CVD (1.23 vs 1.18, P < 0.001). The prevalence of 4 of the top 5 cancers (excluding stomach cancer) will increase regardless of CVD, although these increases will be greater in patients with CVD. The growth rates of urological cancers (prostate and kidney and ureter) with CVD will be significantly higher in men (1.80 vs 1.60, P < 0.001 and 1.35 vs 1.30, P < 0.05, respectively), and those of breast and uterine cancers will be significantly higher in women (1.57 vs 1.21, P < 0.001 and 1.66 vs 1.44, P < 0.001, respectively), compared with the growth rates of patients with each corresponding cancer International Journal of Clinical Oncology (2019) 24:983–994 989 Male Cancer LVSD Urinary Liver bladder 2015 - 2019 (n = 1,722,590) 26.1 17.8 35.3 9.3 18.1 Colon Kidney and Lung and rectum ureter Stomach Prostate 2035 - 2039 (n = 2,060,650) 17.3 13.5 15.0 Liver Leukemia 2015 - 2019 2015-2019 (n = 27,271) 21.8 20352035-2039 - 2039 (n = 34,019) 19.5 14.5 31.1 19.6 Skin Lymphoma 0% 20% 40% 60% LVDD 80% 2015 - 2019 (n = 33,229) 22.3 18.8 13.4 9.1 6.0 0% 19.4 15.8 21.7 9.4 5.4 40% 60% 80% 21.6 17.2 16.9 8.2 8.1 20% 40% 60% AF 80% Kidney Colon Urinary Lung and Others and Prostate Stomach bladder ureter rectum 19.5 26.1 13.7 8.3 6.8 2015 - 2019 (n = 89,476) 20.4 17.5 10.5 10.5 0% 30.2 15.8 40% 60% 80% 23.0 20.8 15.7 9.5 7.2 21.5 16.8 24.6 8.2 4.5 Skin 0% 20% 40% 60% AS 18.2 18.0 100% 2035 - 2039 (n = 14,953) 13.2 11.0 9.8 0% 29.6 18.0 12.8 10.5 10.5 8.2 20% 40% 60% VTE 10.6 2015 - 2019 2015-2019 (n = 18,060) 27.1 18.1 Colon Prostate and rectum 2035 - 2039 2035-2039 (n = 22,730) 24.4 12.5 9.3 6.3 20% 40% 60% NT-proBNP 20152015-2019 - 2019 (n = 36,007) 22.0 80% 16.0 11.6 10.1 9.0 27.2 9.9 7.5 5.9 0% 22.0 14.4 7.4 15.2 8.2 2015 - 2019 2015-2019 (n = 182,697) 19.8 40% 18.8 Prostate 2035 - 2039 2035-2039 (n = 221,481) 22.9 20% 40% 60% 80% 60% 16.4 80% 9.9 6.5 100% 19.3 Colon Urinary and Stomach Lung bladder rectum 29.4 16.6 Leukemia 0% 13.6 Liver Esophagus Colon and Stomach Liver Prostate Lung Esophagus Others rectum 20352035-2039 - 2039 (n = 43,044) 20% CVD 20.8 16.1 Lymphoma Oral cavity and pharynx 100% Leukemia 100% Stomach Lung Esophagus Others Lung Gallbladder and bile duct 0% 80% Oral cavity Lymphoma and pharynx Colon Urinary Leukemia Others and Stomach bladder rectum Prostate 14.9 Lymphoma 80% Liver Lung 2015 - 2019 (n = 11,665) 14.4 Colon and Stomach Prostate Lung Liver Esophagus Others rectum 2035 - 2039 2035-2039 (n = 61,624) 17.1 100% Urinary bladder 2015 - 2019 2015-2019 (n = 53,010) 17.1 13.1 6.3 9.5 20% IHD Colon Urinary and Stomach Liver Lung Others bladder rectum Prostate 2035 - 2039 (n = 108,290) 16.1 17.3 Lymphoma 100% Esophagus 100% 18.9 Oral cavity and pharynx 0% 15.1 Liver 20352035-2039 - 2039 (n = 73,075) 17.7 Others 11.2 8.5 7.0 LVD Kidney Colon Urinary and Stomach Prostate bladder Lung and Others ureter rectum 2035 - 2039 (n = 39,056) 20% 20152015-2019 - 2019 (n = 60,500) 19.7 16.1 Kidney and ureter Esophagus 100% Oral cavity and pharynx 10.7 7.2 Colon Urinary Stomach Lung and bladder rectum Prostate Others 8.2 20.7 12.9 8.7 4.8 Others 19.5 Esophagus Kidney and ureter 100% 0% 20% 40% 60% 80% 100% 13 990 International Journal of Clinical Oncology (2019) 24:983–994 Female Cancer LVSD Thyroid Skin 2015 - 2019 (n = 1,359,090) 30.5 31.8 33.1 18.4 Colon and rectum Others 8.2 11.7 8.0 14.4 2015 - 2019 2015-2019 (n = 6,616) 18.6 Colon and Stomach Uterus Lung rectum Breast 2035 - 2039 (n = 1,591,630) 10.0 9.4 8.1 15.8 Urinary Lymphoma bladder 20352035-2039 - 2039 (n = 7,726) 16.9 16.7 20% 40% 60% LVDD 34.7 15.5 18.2 27.0 80% 14.9 10.4 9.4 7.1 7.0 0% 100% 2035 - 2039 2035-2039 (n = 24,614) 33.1 13.5 13.4 20% 24.8 19.6 20% 40% 60% AF 80% 2035 - 2039 2035-2039 (n = 32,340) 23.2 28.9 0% 8.5 7.1 7.0 5.8 18.6 26.8 11.1 7.04.55.8 19.8 40% 60% 80% 31.7 Colon and rectum 29.6 21.1 20% 40% 60% AS 80% 30.5 0% Breast Ovary Uterus Stomach Others 24.1 8.9 11.5 6.3 5.9 Breast 20352035-2039 - 2039 (n = 14,819) 11.8 9.1 7.8 5.5 13.5 Liver Skin Lung Lymphoma Others 39.5 9.3 40% 60% 35.8 Colon and rectum 32.2 15.3 20% 40% 60% NT-proBNP 20152015-2019 - 2019 (n = 16,435) Uterus 22.2 19.9 Breast 20352035-2039 - 2039 (n = 22,457) 80% 14.6 11.9 7.1 6.9 Colon and Lymphoma Myeloma Ovary rectum 28.0 17.4 13.9 12.1 8.8 5.6 Uterus 0% Fig. 2 (continued) 13 12.4 20% 40% 60% 80% 0% 20.7 20% 40% 12.8 8.4 6.5 9.1 60% 80% 100% Stomach Ovary 2015 - 2019 2015-2019 (n = 70,614) 25.0 22.4 Breast 2035 - 2039 2035-2039 (n = 91,101) 30.4 8.2 7.1 6.7 6.2 19.6 10.5 6.2 5.55.8 Stomach 0% 20% 20.2 Colon and Uterus Lung Lymphoma Others rectum Leukemia 100% 9.9 Kidney and Others ureter Uterus Breast Leukemia CVD Others 10.0 100% Ovary Lymphoma 100% Leukemia 80% 10.8 8.2 7.6 6.9 Stomach Kidney and ureter 0% 10.5 Ovary Lymphoma 2035 - 2039 2035-2039 (n = 18,409) 11.2 8.0 5.6 17.0 20% VTE 2015 - 2019 2015-2019 (n = 15,224) 17.4 10.3 Lung Lymphoma 100% Kidney Stomach and ureter 2015 - 2019 2015-2019 (n = 10,492) 100% 10.0 8.3 6.7 6.6 Lymphoma Gallbladder and bile duct 0% Others Lung Lymphoma 2035 - 2039 2035-2039 (n = 9,851) 17.7 22.0 IHD Others 10.6 11.7 6.9 5.8 17.1 20% 2015 - 2019 2015-2019 (n = 7,781) 17.2 Colon and Lung Uterus Stomach rectum Breast 20352035-2039 - 2039 (n = 28,355) 11.8 8.7 7.8 6.7 19.9 100% Stomach Kidney and ureter 100% Gallbladder Lymphoma and bile duct 20152015-2019 - 2019 (n = 21,985) 80% Colon and Uterus Leukemia Myeloma rectum Breast 16.9 60% Myeloma Kidney and ureter 0% 10.5 Liver Stomach Others 7.6 40% LVD 2015 - 2019 2015-2019 (n = 26,056) 19.5 Colon and Uterus Liver Stomach rectum Breast 7.3 5.8 Urinary bladder Uterus Myeloma Kidney and ureter 2015 - 2019 2015-2019 (n = 19,440) 12.8 Breast Leukemia Ovary Lung Others Thyroid Skin 0% 7.25.0 40% 60% 18.1 Myeloma 80% 100% International Journal of Clinical Oncology (2019) 24:983–994 Table 2 Growth rate of cancer, cancer without CVD and cancer with CVD between 2015–2010 and 2035–2039 in 5 cancers All Cancer Cancer without CVD Cancer with CVD Male Cancer Cancer without CVD Cancer with CVD Prostatic cancer Prostatic cancer without CVD Prostatic cancer with CVD Colon and rectal cancer Colon and rectal cancer without CVD Colon and rectal cancer with CVD Stomach cancer Stomach cancer without CVD Stomach cancer with CVD Lung cancer Lung cancer without CVD Lung cancer with CVD Kidney and ureter cancer Kidney and ureter cancer without CVD Kidney and ureter cancer with CVD Female Cancer Cancer without CVD Cancer with CVD Breast cancer Breast cancer without CVD Breast cancer with CVD Colon and rectal cancer Colon and rectal cancer without CVD Colon and rectal cancer with CVD Uterine cancer Uterine cancer without CVD Uterine cancer with CVD Lung cancer Lung cancer without CVD Lung cancer with CVD Stomach cancer Stomach cancer without CVD Stomach cancer with CVD 991 2015–2019 2035–2039 Growth rate 3,081,680 2,828,369 253,311 3,652,280 3,339,698 312,582 1.19 1.18 1.23 1,722,590 1,539,893 182,697 450,160 414,059 36,101 297,370 263,017 34,353 306,210 276,176 30,034 160,160 142,091 18,069 68,540 62,237 6303 2,060,650 1,839,169 221,481 727,500 662,479 65,021 308,650 271,845 36,805 277,560 248,902 28,658 169,360 150,101 19,259 89,540 81,028 8512 1.20 1.19 1.21 1.62 1.60 1.80 1.04 1.03 1.07 0.91 0.90 0.95** 1.06 1.06 1.07 1.31 1.30 1.35* 1,359,090 1,288,476 70,614 413,970 396,338 17,632 214,900 199,063 15,837 128,240 122,454 5786 110,760 105,780 4980 136,310 131,570 4740 1,591,630 1,500,529 91,101 506,070 478,357 27,713 229,250 211,404 17,846 185,990 176,391 9599 127,010 121,316 5694 129,750 124,745 5005 1.17 1.16 1.29 1.22 1.21 1.57 1.07 1.06 1.13 1.45 1.44 1.66 1.15 1.15 1.14 0.95 0.95 1.06** Odds ratio (95% CI) 1.05 (1.04–1.05) 1.02 (1.01–1.02) 1.13 (1.11–1.14) 1.04 (1.02–1.05) 1.06 (1.04–1.08) 1.01 (0.99–1.03) 1.04 (1.00–1.07) 1.11 (1.10–1.12) 1.30 (1.28–1.33) 1.06 (1.04–1.08) 1.15 (1.11–1.19) 1.00 (0.96–1.04) 1.11 (1.07–1.16) Asterisks indicate statistical differences in CVD prevalence between 2015–2019 and 2035–2039 for each cancer. (P < 0.05, *P < 0.001). The odds ratio indicates the relative risk of CVD comorbidities in 2035– 2039 in comparison with 2015–2019 for each cancer CI confidence interval 13 992 International Journal of Clinical Oncology (2019) 24:983–994 Fig. 3 Sex-, and cancer typespecific projected number of CVDs for the top 10 cancers, 2015–2039. Bar graphs show the number of cancer patients with CVD stratified by 5-year intervals. The top 10 is not a ranking in all cancers but of cancers with CVD in 2015. CVD, cardiovascular disease without CVD. The odds ratio of CVD comorbidity was high in male patients with prostatic cancer (1.13, 95% confidence interval [CI] 1.11–1.14) and in female patients with breast cancer (1.30, 95% CI 1.28–1.33), uterine cancer (1.15, 95% CI 1.11–1.19), and stomach cancer (1.11, 95% CI 1.07–1.16). Fewer female than male patients will have comorbid cancer/CVD; however, the odds ratio of CVD comorbidity will be larger in female than in male patients by 2035 (1.11, 95% CI 1.10–1.12 vs 1.02, 95% CI 1.01–1.02). The projected numbers of sex- and cancer type-specific CVDs in the top 10 cancers between 2015 and 2039 are presented in Fig. 3. 13 Discussion Japan is projected to face an HF pandemic [4, 14, 15] and cancer epidemic [3] in the coming 2 decades owing to an aging population. Therefore, we predicted the future number of cancer patients with CVD. Our study revealed the following new observations: (1) the total number of Japanese cancer patients with CVD was 253,000 in 2015, and is expected to increase rapidly by 30,000 by 2020, peaking at 313,000 in 2030–2034; (2) the CVD population will be predominantly men (2.5-fold the number of women) and ≥ 75 years of age; (3) the growth rate in the number of cancer patients ≥ 75 years will be greater in women than in men; hence, the growth rate in cancer patients with CVD will also International Journal of Clinical Oncology (2019) 24:983–994 be greater in women; and (4) therefore, cancer patients in 2035 will be older and more likely to have CVD than those in 2015, especially women. While cancer patients with CVD will continue to be predominantly male (2.5 times the number of female patients), female patients will experience greater increases in the rate of cancer with CVD. While rates of cancer without CVD will increase 1.16-fold between 2015 and 2035, cancer with CVD rates will increase 1.29-fold. The odds ratio, which is the relative ratio between increasing rates of cancer with vs without CVD, will be 1.11 (Table 2). Breast and uterine cancers show the highest odds ratios at 1.30 and 1.15, respectively. Therefore, healthcare practitioners caring for female (i.e., breast and gynecological) cancers should prepare to treat CVD, especially LVD and VTE. The cancer/CVD epidemic will persist in a decremental phase in Japan after 2005 owing to the aging population. As cancer and CVD are more common in individuals aged over 75 years, cancer centers will be obliged to improve medical care for CVD as well. Cancer patients in 2035 will be older and more likely to have CVD than cancer patients in 2015, especially among women. Japan is projected to face an epidemic of cancer with CVD; as a history of CVD is a strong predictor of relapse or MACE, cancer healthcare practitioners should recognize CVD presence and history. However, the appropriate management of CVD may be deficient owing to unawareness of its existence, failure of patients to mention CVD history (e.g., because of cognitive impairment), communication lapses between hospitals, and shorter medical record storage periods. Electronic medical records containing information on both CVD and cancer should be available at cancer centers. Cancer and CVD incident cases have increased in the US owing to the aging Caucasian population [16, 17]. Cancer patients with CVD are prevalent [5] owing to the longer survival rates of patients with both diseases [18]. The prevalence of HF in patients aged 66 years or older with breast cancer, colorectal cancer, lung cancer, and prostatic cancer were 6.9%, 11.6%, 12.4% and 5.7%, respectively [5]. Although precise comparisons with previously published data are not feasible because of the different methods of assessment and cohort types, our cancer patients may have a lower prevalence of CVD than the US cohort (Supplementary Table 2). However, a future epidemic of cancer with CVD in Japan will likely occur given that Japan has the most rapidly aging population among developed countries [2, 3]. A number of limitations must also be considered. First, this pilot study was retrospective and observational; a prospective study would be preferable for the precise assessment of CVD burden on cancer patients. Second, this was a single-center study; the incidence rates of some cancers differ in Japan (e.g., stomach cancer), and Niigata Cancer Center Hospital may not be a representative Japanese cancer 993 center. However, no single representative hospital has been selected to study the prevalence of cancer with CVD in Japan to date; thus, our hospital is the closest to realizing this goal owing to its suitable population size, average number of cancer patients at each type, and proper diagnosis by cardiologists. Furthermore, the CVD/cancer deaths (ratios) in 2014 in Niigata city and Japan overall were 153/292 (0.524) and 157/294 (0.535), respectively [19]. The ratio in Niigata city was the 15th closest to the overall ratio in Japan, which encompassed 47 prefectures and 21 major cities. CVD and cancer death in Niigata were sufficiently close to the nationwide average that our study can be deemed representative of the country. Third, CVD diagnoses were influenced by access to electrocardiography, echocardiography, vascular echo, CT, and NT-proBNP measurement; the identification of CVD in all cancer patients was not feasible. Moreover, there is a possibility that the actual prevalence of CVD in cancer patients was underestimated because of selection bias arising from the fact that patients with latent CVD in our hospital are not identified simply because of their non-referral to those examinations. Fourth, both our own public data and those of the National Center were based on past-year surveys. With advancing therapies and improved prognoses, future extrapolations may be modified. However, because survival rates in the future promise to improve with advancing anticancer and anti-CVD therapies, the actual number of patients living with cancer and CVD cannot be lower than our projection; in other words, it is highly unlikely that our projection is overestimated. In conclusion, as cancer patients in Japan progressively age, comorbid CVD is expected to increase in prevalence in the near future. Cancer care providers should prepare the medical system for CVD management. The rapid growth of CVD in women with cancer should be recognized, especially in those with breast cancer and gynecological cancers. Acknowledgements We are indebted to Chika Sekine and Tomomi Fujita (cancer registry) for their superb assistance with the datasets; Sumika Ishigaki, Satoko Sakakibara, Chika Yumoto, Megumi Kira, Mitsue Hashidate, Keiko Henmi (echocardiography laboratory) for performing electrocardiography, echocardiography, and vascular echo; Naho Sasaki, Masaki Yoshino, Mayuko Ohtaki, and Naoko Kurematsu (Department of Pharmacology) for pharmacological information; and Dr. Yoshinobu Okada for treating cancer patients with CVD. Compliance with ethical standards Conflict of interest No author has any conflict of interest. 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https://openalex.org/W1968448134	https://europepmc.org/articles/pmc2627675?pdf=render	English	null	B-virus from Pet Macaque Monkeys: An Emerging Threat in the United States?	Emerging infectious diseases	1,998	cc-by	2,918	Dispatches Dispatches B-Virus in Nonhuman Primates Cercopithecine herpesvirus 1 (Herpesvirus simiae or B-virus) frequently infects Old World primates of the genus Macaca. Of at least 19 species of macaques, rhesus, Japanese, cynomol- gus, pig-tailed, and stump-tailed macaques are the species most commonly used in biomedical research (1). Seroprevalence of neutralizing antibodies to B-virus in captive adult macaque populations is 73% to 100% (1-3). Like Herpesvirus simplex virus infection in humans, B-virus infection in monkeys is characterized by lifelong infection with intermittent reactivation and shedding of the virus in saliva or genital secretions, particularly during periods of stress or immunosuppression (4). B-virus infection is transmitted among free-ranging or group-housed animals, primarily through sexual activity and bites. In captivity, as well as in the wild, mature macaques are more likely than immature animals to have been infected with, and shed, the virus. Antibody titer to B-virus indicates infection but can neither confirm nor eliminate actual viral shedding at the time of the bite (4). Most free-ranging monkey populations are thought to be part of the exotic fauna of distant tourist destinations and wild animal parks; however, macaque species have established free- ranging feral populations in Texas and Florida. In such settings, contact between humans and macaques cannot be safely controlled (8-10), and workers and visitors are at risk. Guidelines for B- virus prevention and diagnosis have recently been published (9-12). B-virus from Pet Macaque Monkeys: An Emerging Threat in the United States? Of primary concern when evaluating macaque bites are bacterial and B-virus infections. B-virus infection is highly prevalent (80% to 90%) in adult macaques and may cause a potentially fatal meningoencephalitis in humans. We examined seven nonoccupational exposure incidents involving 24 persons and eight macaques. Six macaques were tested for herpes B; four (67%) were seropositive. A common observation was that children were more than three times as likely to be bitten than adults. The virus must be assumed to be a potential health hazard in macaque bite wounds; this risk makes macaques unsuitable as pets. From 1990 to 1992, 28 U.S. residents reported nonoccupational macaque bites to CDC (L. Chapman, pers. comm.). Since 1993, additional nonoccupational exposure cases have been reported, seven of which (involving 24 persons and eight macaques) are listed in Table 1. Of the six macaques for which herpes B serologic results were available, four (67%) were positive. Two owners refused requests for testing. Four (44%) of nine exposed children were bitten, versus only three (20%) of 12 adults. Children were 3.2 times more likely to be bitten than adults; although a common observation, this association is not statistically significant for this case series. Dispatches Table 1. Selected pet macaque bite casesa,b Primate species, Location age, B-virus status Nature of exposure Comments Illinois Rhesus, 20+ yrs, Household contact Bought at auction, wife bitten B-virus positive (2 adults/3 children), multiple sites, children Cynomolgus, 2-4 yrs, bites, scratches (2 adults) hand-fed monkey B-virus negative Florida Cynomolgus, 2 yrs, Household contact (1 adult), Kissed on lips, ate off owner’s B-virus positive bite (1 child) plate, shared bed Arizona Cynomolgus, 2 yrs, Bites on toe and buttock Unprovoked attack on neighbor, B-virus negative (child) declared vicious animal by judge, no. of household contacts (owner) unreported Cynomolgus, 7 weeks, Household contact Diapered, shared chewed B-virus positive (6 adults), gum, oral ulcers noted by bite on face (1 adult) veterinarian, bite incident at neighborhood bar Macaque, (species Bite on thigh (1 child) Unprovoked attack (climbed undetermined), 2 yrs, fence to bite child) B-virus status unknown Macaque, (species Severe bite (1 child) Injured child attended an and age undetermined), unlicensed day-care facility B-virus status unknown run by monkey owner, 7 other monkeys on premises Minnesota Rhesus, 2 yrs, Household contact, Acquired as “child-substitute” B-virus positive owners’ friend bitten (full-time baby-sitters hired) aCases referred to Centers for Disease Control and Prevention since 1993. bAs of November 1997, no confirmed transmission of B-virus in these persons has been documented. Table 1. Selected pet macaque bite casesa,b tion (1). Recently, acyclovir has prevented progression of the disease in a limited number of patients. In at least three patients, this treatment reversed the neurologic symptoms and was life-saving (7,14,15). Rapid diagnosis and initiation of therapy are of paramount impor- tance in preventing death or permanent disability in surviving patients. and breeders of nonhuman primates agree that as a rule, all these animals bite (16,17). Biting incidents eventually bring the animals to the attention of animal control authorities. Most state health departments can require that any biting nondomestic animal be euthanized and the brain be submitted for rabies testing. B-Virus in Humans Symptomatic human infection with B-virus is rare; fewer than 40 cases were reported from 1933 to 1994 (1,4-7,13-15; CDC, unpub. data). However, the consequences of symptomatic infection may be severe. Viral infection rapidly progresses to central loci in the spinal cord and, eventually, the brain. Of 24 known symptomatic patients whose cases were reviewed in 1992, 19 (79%) died (CDC; unpub. data). B-virus disease in humans usually results from macaque bites or scratches (4). Incubation periods may be as short as 2 days, but more commonly are 2 to 5 weeks (1,3,5-7; Centers for Disease Control and Prevention [CDC], unpub. data). Most documented infections have occurred among biomedical research employees who had occupational exposure to macaques, although transmission has also been documented among laboratory workers handling infected central nervous system and kidney tissues (1,5). Before 1987, most surviving human patients had moderate to severe neurologic impairment, sometimes requiring lifelong institutionaliza- 117 Vol. 4, No. 1, January–March 1998 Emerging Infectious Diseases Emerging Infectious Diseases Dispatches Dispatches Dispatches Table 2. Federal regulations regarding nonhuman primates Agency Statute Regulation Subjects Departments of Health, Public Health Services 42 CFR 71.53 Importation, distribution, bona fide Centers for Disease Act, 42 USCS 201 uses in the U.S., breeding colony Control and Prevention requirements, pet/avocationist uses Department of Animal Welfare Act, 9 CFR Licenses (breeders, dealers, laboratories, Agriculture 7 USCS 2131-2159 Subchapter A exhibitors, auctions), interstate health certificates, humane care and transport Department of the Endangered Species 50 CFR 10, 11, Endangered species, smuggling, Interior, U.S. Fish Act, 16 USCS 1540 13, 14, 16 interstate sales and Wildlife Service Lacey Act, 18 USCS 42 Table 2. Federal regulations regarding nonhuman primates in 33% of cases (20,21). level educational programs, or full-time zoologic exhibition. Furthermore, the regulation states, “the maintenance of nonhuman primates as pets, hobby, or an avocation with occasional display to others is not a permissible use” (18). in 33% of cases (20,21). level educational programs, or full-time zoologic exhibition. Furthermore, the regulation states, “the maintenance of nonhuman primates as pets, hobby, or an avocation with occasional display to others is not a permissible use” (18). In 1990, the American Veterinary Medical Association issued a general policy statement opposing the keeping of wild animals (especially those inherently dangerous to humans) as pets and advising veterinarians to exert their influence to discourage this practice (22). In 1995, updated guidelines for the prevention and treatment of B-virus infections in exposed persons were published (12). Despite these continuing public health educational efforts, nonhuman primates (including macaques) con- tinue to be marketed and kept as pets in many states (16,17,23). All states require their citizens to comply with applicable federal regulations. Many state officials, however, may be unaware of regulatory restrictions on the uses and distribution of nonhuman primates and may be confused by the distinctions among federal agencies regarding regulatory restrictions on captive-bred animals. State wildlife authorities may not know that a federal public health regulation prohibits the keeping (“maintenance”) of nonhuman primates imported after October 10, 1975, as pets, for a hobby, or as an avocation; likewise, many do not know the compelling public health and safety reasons for enforcement. Human and Macaque Interactions Most owners form an emotional bond with infant primates. This bond is probably strength- ened by the neonatal monkey’s physical and behavioral resemblance to a human infant. Although physically and emotionally dependent on their mothers (or human substitutes) for up to 2 years of age, most macaques exhibit unpredictable behavior as they mature. Males tend to become aggressive, and both male and female macaques bite to defend themselves and to establish dominance. Dominance within the social hierarchy of macaques is established by aggression toward other monkeys, generally the younger and smaller members of the group. Both veterinary specialists Table 2 lists the principal federal regulations affecting the possession, distribution, and uses of nonhuman primates. The United States is obligated under the Convention in International Trade in Endangered Species (CITES) to restrict and control trafficking in exotic and endangered species. Since October 10, 1975, U.S. Public Health regulation 42 CFR 71.53(c) has prohibited the importation of nonhuman primates into the United States as pets, and neither nonhuman primates imported since that date nor their offspring may be legally bred or distributed for any uses other than bona fide science, university- 118 Vol. 4, No. 1, January–March 1998 Emerging Infectious Diseases References 1. Palmer AE. B virus, Herpesvirus simiae: historical perspective. J Med Primatol 1987;16:99-130. 2. Shah KV, Morrison JA. Comparison of three rhesus groups for antibody patterns to some viruses: absence of active simian virus 40 transmission in the free- ranging rhesus of Cayo Santiago. Am J Epidemiol 1972;99:308-15. The public resources deployed when a monkey-bite case is referred to public health authorities are similar to those required for rabies investigations (M. Leslie and T. Parrott, unpub. obs.). Persons bitten by pet and feral macaques are more likely than persons bitten in the workplace to require public resources, delay seeking medical care, and have an initial medical evaluation by care givers who are largely unfamiliar with the potentially serious conse- quences of B-virus exposure (23). In contrast, occupational exposure generally occurs within highly structured workplace settings, where health professionals are prepared to provide prompt, appropriate, and specific care at no public cost. 3. Orcutt RP, Pucak GJ, Foster HL, Kilcourse JY. Multiple testing for the detection of B virus antibody in specially handled rhesus monkeys after capture from virgin trapping grounds. Lab Anim Sci 1976;26:70-4. 4. Weigler BJ. Biology of B Virus in macaque and human hosts: review. Clin Infect Dis 1992;14:555-67. 5. Hummeler K, Davidson WL, Henle W, LaBoccetta AC, Ruch HG. Encephalomyelitis due to infection with Herpesvirus simiae (Herpes B virus): report of two fatal laboratory acquired cases. N Engl J Med 1959;261:64-8. 6. Centers for Disease Control. B-virus infections in humans—Michigan. MMWR Morb Mortal Wkly Rep 1989;38:453-4. 7. Holmes GP, Hilliard JK, Klontz KC, Rupert AH, Schindler CM, Parrish E, et al. B-virus (Herpesvirus simiae) infection in humans: epidemiologic investigations of a cluster. Ann Intern Med 1990;112:833-9. Ongoing efforts to establish B-virus–free macaque colonies illustrate the difficulties of ascertaining B-virus–negative status, even with a battery of sophisticated laboratory tests and extended longitudinal follow-up of individual macaques (25). The high percentage of death in known cases of human B-virus disease under- scores the potential seriousness of all bite or scratch exposures from macaques. 8. Centers for Disease Control. Guidelines for prevention of Herpesvirus simiae (B-virus) infection in monkey handlers. MMWR Morb Mortal Wkly Rep 1987;36:681- 2, 687-9. 9. The B Virus working group. Guidelines from prevention of Herpesvirus simiae (B virus) infection in monkey handlers. J Med Primatol 1988;17:77-83. 10. Hilliard JK. 1990-1994 yearly comparisons; B-virus Resource Laboratory. San Antonio (TX): Southwest Foundation for Biomedical Research; 1995. Stephanie R. Ostrowski,* Mira J. Leslie,† Terri Parrott,‡ Susan Abelt,¶ and Stephanie R. Ostrowski,* Mira J. Leslie,† Terri Parrott,‡ Susan Abelt,¶ and Patrick E. Piercy§ *Centers for Disease Control and Prevention, Atlanta, Georgia, USA; †Arizona Department of Health Services, Phoenix, Arizona, USA; ‡Pembroke Park Animal Hospital, Pembroke Park, Florida, USA; ¶Lake Superior Zoological Gardens, Duluth, Minne- sota, USA; and §Illinois Department of Public Health, Springfield, Illinois, USA References The extremely high prevalence of B-virus along with their behavioral characteristics make the macaque species unsuitable as pets. 11. Wells DL, Lipper SL, Hilliard JK, Stewart JA, Holmes GP, Herrmann KL, et al. Herpesvirus simiae contamination of primary rhesus monkey kidney cell cultures. CDC recommendations to minimize risks to laboratory personnel. Diagn Microbiol Infect Dis 1989;12:333-6. Some Public Health Consequences of the Nonhuman Primate Pet Trade The pet trade in a variety of nonhuman primate species, and particularly the apparent increase in macaque species as part of this trade, may constitute an emerging infectious disease threat in the United States. Although the U.S. Fish and Wildlife Service indicates that illegal traffic in nonhuman primates is a significant aspect of the estimated $3 billion worth of wildlife illegally traded in the United States annually, more data are needed on the actual number of macaques in the private sector and on trends in the population (24; U.S. Fish and Wildlife special agents, pers. comm.). ¶Lake Superior Zoological Gardens, Duluth, Minne- sota, USA; and §Illinois Department of Public Health, Springfield, Illinois, USA The Frequency of Exposure Resulting in Infection Much remains to be learned about the pathogenesis of B-virus infection in humans. In this very limited case series (Table 1), one family (two adults and two of three children) exposed to a B-virus positive macaque had flulike symp- toms. One of the adults had additional symptoms related to the injury site, which suggested B- virus infection. In the other six cases, no suspect clinical symptoms were noted, and disease- specific antiviral postexposure prophylaxis was not given. B-virus is still rare, and diagnostic evaluation of clinical cases of aseptic meningitis does not routinely include B-virus testing. Captive-bred offspring of animals purported to have been imported before October 10, 1975, are frequently offered for sale. Without documentation it is very difficult to determine whether this is the case. Depending on the specific circumstances, it is possible for undocumented animals to be consid- ered deliberately misclassified (i.e., intentionally mislabeled), a violation under the Lacey Act (18 USC 42) and under 16 USC 3373 (19). In 1987 and 1988, occupational safety guidelines were published based on evidence that all macaque species are inherently dangerous to humans because of the risk for B-virus transmission, as well as the likelihood of serious physical injury from bite wounds (9-12,14,15). Several recent reviews of monkey-bite injuries worldwide indicate that severe lacerations, wound infections, and permanent sequelae (e.g., flexure contractures, osteomyelitis) were present Owners of pet macaques are often reluctant to report bite injuries from their pets, even to their medical care providers, and may fail to appreciate that the premonitory headache and flulike symptoms (which may lead them to seek medical attention) could be associated with healed, often minor, bite wounds dating back more than a month (23). The Southwest Foundation for 119 Vol. 4, No. 1, January–March 1998 Emerging Infectious Diseases Dispatches Biomedical Research, which is the designated National Institutes of Health B-virus resource laboratory, reports processing 2,000 to 3,000 human diagnostic specimens per year between 1990 and 1994, or approximately 200 per month, most of which reflect occupational exposure (8). Biomedical Research, which is the designated National Institutes of Health B-virus resource laboratory, reports processing 2,000 to 3,000 human diagnostic specimens per year between 1990 and 1994, or approximately 200 per month, most of which reflect occupational exposure (8). Francis, Mr. C. Langkop; Drs. R. Martin, J. Hilliard, D. Watkins; Capt. J. Thompson; Special Agents P. Bosco, D. Burleson, J. English, S. Hamilton, T. Karabinoff, D. Kirkby, D. Manera, G. Phillips, G. Phocas, T. Santelle, J. Sommers, C. Tabor; Dr. J. Cheek; and the Division of Viral and Rickettsial Diseases, Animal Resources Branch, and the Division of Quarantine, CDC. Acknowledgments The authors thank Drs. D. Manning, S. O’ Marro, R. Montrey, T. Burke, D. Morton, and J. Thulin; Mr. J. 120 Emerging Infectious Diseases Vol. 4, No. 1, January–March 1998 Dispatches Dispatches 12. Holmes GP, Chapman LE, Stewart JA, Straus SE, Hilliard JK, Davenport DS, et al. Guidelines for the prevention and treatment of B-virus infections in exposed persons. Clin Infect Dis 1995;20:421-39. 19. Anderson RS. Lacey Act. Case law update and current issues in federal wildlife prosecution. Washington: U.S. Department of Justice, Environment and Natural Resources Division; 1993. 20. Goldstein EJC, Pryor EP, Citron DM. Simian bites and bacterial infection. Clin Infect Dis 1995;20:1551-2. 13. Artenstein AW, Hicks CB, Goodwin BS, Hilliard JK. Human infection with B virus following a needlestick injury. Reviews of Infectious Diseases 1991;13:288-91. 21. Janda DH, Ringler DH, Hilliard JK, Hankin RC, Hankin FM. Nonhuman primate bites. J Orthop Res 1990;8:146-50. 14. Centers for Disease Control. B-virus infection in humans—Pensacola, Florida. MMWR Morb Mortal Wkly Rep 1987;36:289-90, 295-6. 22. American Veterinary Medical Association. Policy Statement on Wild Animals as Pets. In: 1995 AVMA Directory. Schaumburg (IL): The Association; 1995. 15. Centers for Disease Control. B-virus infection in humans—Michigan. MMWR Morb Mortal Wkly Rep 1989;38:453-4. 23. Paulette J. “Yes, monkeys will bite!” The Simian 1996 Feb;6-8. 16. Hoctor P. Short hints on raising exotics. Animal Finders Guide 1995 Sep 1;12:44-5. 24. Webster D. The looting and smuggling and fencing and hoarding of impossibly precious, feathered and scaly wild things: inside the $10 billion black market in endangered animals. The New York Times Magazine 1997 Feb 16: 27-33, 48-50. 17. Johnson-Delaney CA. The pet monkey: health care and husbandry guidelines. Journal of Small Exotic Animal Medicine 1991;1:32-7. 18. Centers for Disease Control. Import restrictions on nonhuman primates. Atlanta (GA): Centers for Disease Control; 1982 Nov 17. DQ-CPS Advisory Memorandum No. 65. 25. Hilliard J. Managing macaques and herpes B. Presented at 4th National Symposium on Biosafety: Working Safely with Research Animals; 1997 Jan 27-31; Atlanta, Georgia. 121 Emerging Infectious Diseases Emerging Infectious Diseases Vol. 4, No. 1, January–March 1998
W2802659325.txt	https://revues.cirad.fr/index.php/REMVT/article/download/31528/31247	fr		Caractérisation zootechnique et formule barymétrique de la race zébu Azawak à Ménaka au nord du Mali	Revue d'élevage et de médecine vétérinaire des pays tropicaux/Revue d'élevage et de médecine vétérinaire des pays tropicaux	2,018	cc-by	4,605	Mots-clés Résumé Bos indicus, bovin Azawak, croissance, performance de reproduction, poids corporel, Mali L’objectif de cette étude était de participer à la sauvegarde et à l’amélioration des performances du zébu Azawak au Mali en proposant une estimation des performances de croissance et de reproduction de cette race bovine élevée au nord du pays. Le suivi a concerné 1129 animaux dans quatre communes du cercle de Ménaka, entre novembre 2009 et février 2010. A 30 jours, les mâles pesaient en moyenne 39 ± 10 kg et les femelles 32 ± 4 kg. Aucune différence significative (p > 0,05) n’a été enregistrée entre les poids adultes des taureaux (370 ± 88 kg) et des vaches (327 ± 46 kg). L’âge au premier vêlage a été de 50 ± 11 mois avec un intervalle entre vêlages de 16 ± 4 mois. Les corrélations du poids avec le périmètre thoracique (r = 0,95) et la hauteur au garrot (r = 0,94) ont été les plus élevées. Des équations barymétriques ont été établies pour chaque catégorie animale (veau/velle, génisse/taurillon, vache/taureau) au moyen de régressions polynomiales du poids (y) sur le périmètre thoracique (x). Celles-ci pourront servir à établir une table de conversion du périmètre thoracique en poids vif ou un ruban barymétrique à l’attention des agents de l’élevage, afin de faciliter un suivi zootechnique et sanitaire des animaux sur le terrain. Submitted: 26 May 2016 Accepted: 1 February 2018 Published: 23 April 2018 DOI : 10.19182/remvt.31528 ■ Comment citer cet article : Touré A., Antoine-Moussiaux N., Kouriba A., Leroy P., Moula N., 2017. Zoo- technical characterization and barymetric formula of the Azawak zebu breed in Menaka, Northern Mali. Rev. Elev. Med. Vet. Pays Trop., 70 (4): 115-120, doi: 10.19182/remvt.31528 ■ INTRODUCTION Dans le nord du Mali, l’élevage bovin est soumis à des contraintes climatiques (aridité) et socioéconomiques (accès à l’eau potable et abris de nuit, parc de vaccination, marché à bétail) freinant son essor. De plus, dans un milieu aride, où la ressource alimentaire évolue fortement au cours de l’année, les éleveurs sont en quête permanente de satisfaction des besoins alimentaires des animaux (petite transhumance, séjour sur terres salées). En fin de saison sèche, les animaux 1. Fundamental and Applied Research for Animal and Health (FARAH), Productions animales durables, Faculté de médecine vétérinaire, Université de Liège, Quartier Vallée 2, avenue de Cureghem 6, 4000 Liège, Belgique. 2. Institut vétérinaire tropical, Faculté de médecine vétérinaire, Université de Liège, Liège, Belgique. 3. Institut d’économie rural (IER), Bamako, Mali. * Auteur pour la correspondance Tél. : +32 43 66 41 24 ; fax :+32 43 66 41 22 Email : Nassim.Moula@ulg.ac.be https://creativecommons.org/licenses/by/4.0/ sont très amaigris, ce qui affecte la viande en termes de flaveur et de qualité. Des études ont été menées au Niger, en Côte d’Ivoire et au Burkina Faso pour évaluer les performances de production de la race Azawak (Dodo et al., 2001 ; Marichatou et al., 2005 ; Sokouri et al., 2010). Cependant, rares sont celles qui ont concerné la race zébu Azawak au Mali. La race Azawak est à présent de plus en plus métissée avec les races Peuhle et M’bororo venant des cercles d’Ansongo et de Gao au Mali, voire du Burkina Faso ou du Niger. C’est donc pour faire face à la disparition de cette race bovine locale présentant d’intéressantes caractéristiques en termes de rusticité, d’adaptation et de production que le gouvernement malien a mis en œuvre le Projet d’appui à la sélection et à la multiplication du zébu Azawak au Mali (PASMZAM) visant à conserver la race Azawak. La présente étude s’inscrit dans ce programme et vise à caractériser le système d’élevage de la race Azawak, à déterminer ses caractéristiques de croissance et de reproduction dans son milieu d’élevage, ainsi qu’à déterminer une formule barymétrique permettant d’estimer facilement le poids vif des animaux sur le terrain. ■ PRODUCTIONS ANIMALES ET PRODUITS ANIMAUX Abdoulaye Touré 1 Nicolas Antoine-Moussiaux 1, 2 Aly Kouriba 3 Pascal Leroy 1, 2 Nassim Moula 1, 2 * Revue d’élevage et de médecine vétérinaire des pays tropicaux, 2017, 70 (4) : 115-120 Caractérisation zootechnique et formule barymétrique de la race zébu Azawak à Ménaka au nord du Mali 115 Zootechnical characterization of the Azawak zebu breed in Mali Revue d’élevage et de médecine vétérinaire des pays tropicaux, 2017, 70 (4) : 115-120 ■ PRODUCTIONS ANIMALES ET PRODUITS ANIMAUX ■ MATERIEL ET METHODES 116 Zone d’étude L’étude a été réalisée dans la région de Gao (cercle de Ménaka), située dans la vallée fossile de l’Azawak (figure 1). Le relief de la commune est essentiellement constitué de dunes de sables, de vallées, de basfonds et de plaines. Le climat se caractérise par l’alternance d’une longue saison sèche (huit à neuf mois) et d’une courte saison des pluies (quatre mois de juin à septembre) avec une forte variabilité interannuelle. C’est une zone aride avec une pluviométrie annuelle comprise entre 100 et 250 millimètres et des températures maximales moyennes élevées, plus de 40 °C en avril et mai. La végétation est de type désertique où prédominent les épineux du genre Acacia spp. Dans la strate herbacée prédominent les graminées annuelles comme Aristida mutabilis, Cenchrus biflorus et Eragrostis tremula. Modes d’élevage Dans cette région, et plus particulièrement dans le cercle de Ménaka, le zébu Azawak est le principal capital pour les communautés touarègues. L’Azawak est soumis à un élevage de type extensif, caractérisé par une longue période de mobilité (octobre à juin) et une population contrainte à une agriculture de subsistance (Beeler, 2006). Les animaux se déplacent sur les vastes pâturages naturels de la zone sahélienne sous la conduite d’un gardien à la recherche de ressources alimentaires et d’eau (Turner et al., 2014). La transhumance et le nomadisme sont les deux modalités générales de ces déplacements (Beeler, 2006). La culture du bourgou (Echinochloa stagnina) visant à affourager les bovins est pratiquée aux abords du fleuve et des grands lacs permanents. L’importance de l’élevage au nord du Mali a poussé le gouvernement à lancer plusieurs programmes de vulgarisation associés à des projets de développement local basés sur les productions animales (bovin et dromadaire) et végétales (mil, sorgho et riz). Echantillonnage L’étude a concerné cinq communes du cercle de Ménaka. Pour inclure un élevage (selon les termes du PASMZAM) l’éleveur devait a) être reconnu comme sélectionneur de la race zébu Azawak, et b) avoir un troupeau résidant dans la zone d’étude. Au total 1129 bovins dont 67 % de femelles ont été concernés par l’étude. Elevages bovins Les vaches allaitantes et leurs veaux sont gardés à la ferme et conduits par de jeunes bouviers. Les veaux et velles sont attachés la nuit pour les empêcher de téter leur mère pour les besoins de la traite. Le surplus de lait produit par les vaches en lactation qui ne font pas la transhumance (20 %) est autoconsommé ou vendu pour les besoins de trésorerie. Les veaux sont mis au pâturage lorsqu’ils atteignent l’âge de trois à quatre mois. En saison sèche, les troupeaux broutent les résidus de récoltes. Ces zones constituent l’essentiel du territoire pastoral. Les résidus de récoltes constituent une source de biomasse non négligeable pour les bovins. Les pratiques locales admettent qu’après les récoltes l’espace champêtre soit ouvert à la pâture. La vente des résidus et des tas de bourgou aux éleveurs procure des revenus aux producteurs. Après épuisement des résidus, ils sont menés dans les bourgoutières. En saison des pluies, les animaux sont dans les pâturages des zones exondées avec un minimum de main-d’œuvre. Le pouvoir traditionnel est l’interlocuteur principal et incontournable dans les actions de décisions et des règles foncières qui en découlent. Figure 1 : carte du Mali montrant la zone d’étude. Caractérisation zootechnique de la race zébu Azawak au Mali Suivi des élevages bovins Dès l’installation du projet, chaque animal était bouclé et accompagné d’une fiche individuelle de suivi sur la période 2001–2010. Des informations d’identification relatives à chaque animal (numéro, date de naissance, sexe, rang de mise bas et site) ont été collectées lors d’une visite. Les pesées étaient réalisées tôt le matin avant le départ des animaux au pâturage. Lors des pesées, les animaux traversaient d’abord le couloir de contention du parc de vaccination sur une bascule pèse-bétail d’une capacité de 1500 kg et d’une précision de 1,5 kg. Une bascule mobile d’une portée maximale de 300 kg avec une précision de 500 g a été affectée aux jeunes. Deux toises graduées en centimètres et des rubans métriques (de 1,5 m et 3 m) ont servi pour les mensurations des animaux (périmètre thoracique, hauteur au garrot et longueur scapulo-ischiale de la pointe de l’épaule à la pointe du bassin). Le périmètre thoracique et la hauteur au garrot (distance verticale entre le sol et le sommet du garrot, immédiatement en arrière de la bosse) ont été retenus pour établir la barymétrie. Analyses statistiques Le modèle de prédiction du poids (statistique descriptive, équation de régression) a été estimé en utilisant le logiciel R (version 3.0.1) (Coghlan, 2011). Les effets du sexe et de l’âge ont été étudiés au départ d’une analyse de la variance (Anova). La modélisation de la courbe de croissance des bovins a été réalisée selon SAS (Statistical Analysis System, 2001) à des âges type d’après l’équation de Gompertz (France et al., 1996 ; Martin, 1967) : Y = α × exp(-ß × exp[-γ × t]) où Y est le poids des animaux (en kg), α le poids asymptotique (en kg), ß la constante d’intégration, γ le paramètre de vitesse de croissance (facteur de maturation) et t l’âge (en mois). L’âge à l’inflexion (t) correspond à la période où la croissance est maximale et est calculé par la formule t = (1/ γ) × ln│ß│ où γ correspond au poids à l’âge t (Porter 2010). Ces paramètres ont été estimés par la procédure de la régression non linéaire selon la méthode Marquardt (Proc nln, SAS, 2001). L’équation de prédiction du poids a été déterminée à partir des différents paramètres morphologiques mesurés en appliquant la procédure statistique Stepwise (Linear regression, proc reg, option stepwise). ■ RESULTATS Caractérisation des exploitations et des troupeaux Parmi les éleveurs enquêtés 83 % pratiquaient l’élevage comme activité unique, et 17 % combinaient l’élevage et l’agriculture de subsistance. L’effectif moyen de bovins a été de 12 ± 6 têtes (médiane 16 ; min–max 3–31). L’âge moyen des taureaux a été de 4 ans et 2 mois ± 1 an et 5 mois, alors que chez les vaches il a été de 6 ans et 7 mois ± 1 an et 6 mois. Les adultes de plus de trois ans d’âge représentaient 46 % de l’effectif, dont 39 % étaient des vaches laitières en production et 7 % des taureaux. A partir de six ans les mâles représentaient 1 % de l’effectif et les femelles 26 %. Performances Croissance La figure 2 présente les courbes de croissance par sexe. A 12 mois (veau/velle), les poids moyens des mâles et des femelles ont été respectivement de 118 ± 25 kg et 113 ± 36 kg. A 2 ans et 8 mois (taurillons), Figure 2 : courbe de croissance de la race Zébu Azawak estimée par l’équation de Gompertz dans les conditions d’élevage extensif à Ménaka au Mali. et 3 ans et 4 mois (génisses) ces valeurs ont été respectivement de 274 ± 57 kg et 242 ± 52 kg. A ce stade le gain moyen quotidien a été de 245 ± 77 g pour les mâles et de 214 ± 83 g pour les femelles. La croissance observée chez les jeunes entre 3 et 9 mois a été respectivement pour les mâles et les femelles de 219 ± 42 g/j et de 199 ± 53 g/j. La courbe de croissance a été différente selon le sexe (p < 0,01). Les paramètres relatifs à la courbe de croissance de Gompertz diffèrent entre les sexes (Dibanzilua et al., 1995). Le poids asymptotique (α) ainsi que la constante d’intégration (β) des mâles ont été supérieurs à ceux des femelles, respectivement de 438 contre 363, et de 2,4 contre 2,3. Les paramètres de la courbe de croissance estimés par l’équation de Gompertz sont : [-0,0442 t] ) Equation mâle, Y = 437,8e (-2,36e [-0,0508 t]) Equation femelle, Y = 363,4e (-2,34e Reproduction L’âge moyen au premier vêlage, déterminé chez 363 primipares, a été de 50 ± 11 mois (médiane 48). Il a été entre 33 et 40 mois chez 14 % des animaux (tableau I). L’âge minimum au premier vêlage signifie une saillie fécondante à 20 mois. L’intervalle moyen entre vêlages a été de 16 ± 5 mois avec des extrêmes allant de 10 à 31 mois et une médiane de 15 mois. L’intervalle entre vêlages (IVV) a varié d’un an à un an et demi chez 53 % des vaches. Equation de régression du poids Le périmètre thoracique a été retenu pour la détermination de formules barymétriques en raison de sa corrélation plus élevée avec le poids (r = 0,95) par rapport à la hauteur au garrot. Chez les mâles et les femelles, le poids prédit avec la régression polynomiale de y sur x à deux degrés était très proche du poids observé (tableau II). Le coefficient de détermination du modèle de régression du poids des taureaux a été de 84 %. Tableau I Age moyen au premier vêlage et intervalle entre vêlages des zébus Azawak à Ménaka au Mali n Moyenne Ecart-type Médiane Minimum Maximum Age au premier vêlage (mois) Intervalle entre vêlages (mois) 363 50,1 11,2 47,9 33,1 94,2 164 16,2 4,5 15,4 12,2 18,2 Revue d’élevage et de médecine vétérinaire des pays tropicaux, 2017, 70 (4) : 115-120 Méthode 117 Zootechnical characterization of the Azawak zebu breed in Mali Revue d’élevage et de médecine vétérinaire des pays tropicaux, 2017, 70 (4) : 115-120 ■ PRODUCTIONS ANIMALES ET PRODUITS ANIMAUX Tableau II 118 Equations de régression par catégorie d’âge des zébus Azawak à Ménaka au Mali Age Equation de régression R2 Sy < 1 an Y = 0,024997X2 – 2,612304X + 93,177691 94 12,32 Taurillon et génisse 1–3 ans n = 279 Y = 0,01899X2 – 1,912098X + 101,335727 Y = 2,6942X – 174,0277 78 57 18,22 24,7 Vache n = 379 Y = 0,038932X2 – 8,243556X – 640,534367 55 35,45 Taureau Y = 0,036282X2 – 5,740076X + 309,491414 n = 175 84 44,81 Veau et velle n = 296 > 3 ans Y : poids estimé (kg) ; X : périmètre thoracique (cm) R2 : coefficient de détermination ; Sy : écart-type résiduel (kg) ■ DISCUSSION Performance de reproduction et de croissance La distribution globale des paramètres étudiés présentée aux figures 3 et 4 indique que plus de la moitié des bovins avaient un intervalle entre vêlages compris entre 12 et 18 mois. L’âge au premier vêlage et l’IVV calculés ont été largement plus élevés que ceux observés chez le bovin zébu Azawak dans son berceau au Niger (33,6 ± 1,0 mois et 11,9 ± 2,2 mois ; Achard et Chanono, 2006) mais en deçà par exemple de la race Boran en Ethiopie (57,6 ± 2,4 mois et 20,7 ± 0,5 mois ; Denbarga et al., 2012). Habib et al. (2010), et Kibwana et al. (2012) suggèrent que des différences d’âge au premier vêlage et d’IVV sont d’ordre physiologique, sanitaire ou relatives à la conduite de la mise à la reproduction. L’IVV (487 ± 133 jours) a été plus long que celui obtenu chez le zébu Gobra au Sénégal (473,2 ± 7,8 jours ; Denis, 1971) et chez le zébu Azawak au Niger (432 jours ; Achard et Chanono, 2006). Cette différence pourrait être liée à la mesure pratiquée sur un troupeau en station où les conditions d’élevage sont meilleures que celles en milieu paysan (Boujenane et Aissa, 2008 ; Denbarga et al., 2012). Quant au gain moyen quotidien, les valeurs observées ont été élevées en comparaison avec celles obtenues par Youssao et al. (2000) chez la race Borgou au Benin qui étaient de 224 g et 201,7 g respectivement pour les mâles et les femelles de 3–6 mois. Ce résultat reste inférieur au gain moyen de poids de 500 g observé chez le zébu Azawak à la station de Toukounous au Niger (Achard et Chanono, 1995) et de 306 g chez le zébu Goudali au Burkina Faso (Marichatou et al., 2005). Le pourcentage de femelles âgées de six ans et plus (26 % de l’effectif des vaches) indique un regain d’intérêt des éleveurs pour cette race ; au Niger seulement 6 % de l’effectif avait au moins douze ans (Achard et Chanono, 1995). Après la sécheresse des années 1980–1985 la race Azawak était en voie de disparition dans cette zone du Mali mais en la sélectionnant sur les critères phénotypiques et en évitant son croisement avec d’autres races qui transhument dans la zone elle a commencé à s’imposer. L’équation de Gompertz utilisée dans cette étude a montré un âge d’inflexion de la croissance respectivement à 19 et 17 mois chez les mâles et les femelles. Les femelles du groupe d’analyse ont présenté le poids asymptotique le plus faible (363 kg). Cet âge indique le moment où les animaux atteignent leur croissance maximale (Porter et al., 2010). C’est l’âge idéal où le rapport coût de l’alimentation / croissance est optimal, et où il est conseillé de garder ou de sortir les animaux du lot de testage (Poivey et al., 1980). Choix de formules de prédiction pour la barymétrie Figure 3 : distribution par tranches d’âges des fréquences des intervalles entre vêlages chez le zébu Azawak à Ménaka au Mali. Figure 4 : distribution de fréquence des âges à la première mise bas chez le zébu Azawak dans le cercle de Ménaka. La détermination rapide et facile du poids vif des bovins est souvent nécessaire pour faire face à certaines exigences techniques comme la détermination des besoins d’entretien des animaux et le contrôle de l’aptitude à la production de viande (Akouango et al., 2010 ; Symoens et Hounsou-Ve, 1991). La valeur du coefficient de détermination (R2) obtenue par l’utilisation de la régression multiple reste faible pour les vaches (0,55). Selon Symoens et Hounsou-Ve (1991), les formules linéaires sembleraient être plus adaptées aux jeunes dont le poids vif se situe entre 150 et 250 kg. Au-delà de ces limites, elles tendraient à le sous-estimer. Cela revient à dire que plusieurs facteurs comme l’âge et l’état physiologique de l’animal interviennent dans l’estimation du poids vif des bovins (Akouango et al., 2010). De toutes les mesures et équations proposées, il apparaît clairement que le périmètre thoracique est le meilleur estimateur du poids (Kashoma et al., 2011 ; Poivey et al., 1980). Des droites de régression fractionnée, établies pour des intervalles de variation du poids correspondant aux différentes catégories animales (Denis, 1971 ; Nicholson Caractérisation zootechnique de la race zébu Azawak au Mali et Sayers, 1987), permettent de suivre toutes les phases de croissance bovine. Elles sembleraient être pratiques dans les conditions d’élevage extensif notamment du Sahel pour des raisons liées aux déplacements fréquents et à la contention des animaux. Poivey et al. (1980) ont utilisé la liaison de curvilinéarité entre le poids et le tour de poitrine en les comparant avec des droites de régression linéaire et multiple qui tenaient compte de la mesure de la hauteur au garrot. Elles ont montré plus de précision au regard de leur R2 observé. Dans l’ensemble, l’erreur dans l’estimation du poids par les équations linéaires s’avère plus importante que celle des régressions polynomiales proposées. ■ CONCLUSION La modélisation de la croissance par l’équation de Gompertz a permis de choisir des animaux pour le testage. Le périmètre thoracique, paramètre facilement mesurable, a constitué un élément estimant le mieux le poids des animaux. Bien que les animaux aient été conduits dans un environnement non contrôlé (suivi vétérinaire et zootechnique approprié), les résultats de l’évaluation des performances de reproduction relatives à l’âge au premier vêlage et à l’intervalle entre vêlages ont révélé que la race locale zébu Azawak présentait de bonnes aptitudes pour l’amélioration de la productivité d’un élevage. A cet effet, elle constitue un atout considérable pour la valorisation et le développement de l’élevage bovin au Mali. Les différentes formules barymétriques établies en fonction du sexe et de l’âge chez le zébu Azawak fournissent une précision suffisante pour un suivi pondéral plus rapide et fréquent sur un nombre d’animaux plus élevé que par un système de pesage classique. Pour la mise en œuvre d’un programme de sélection basé sur les performances de croissance, il faudra alors choisir les animaux dont l’âge à l’inflexion (croissance maximale) soit de 19 mois pour un poids de 161 kg chez les mâles contre 17 mois pour un poids de 134 kg chez les femelles. REFERENCES Achard F., Chanono M., 2006. Exemple d’une gestion pastorale réussie au Sahel : la station d’élevage de Toukounous (Niger). Sécheresse, 17 (1-2) : 76-82 Akouango F., Ngokaka F., Monpoundza C.P., Emmanuel K., 2010. Barymetrie formulas and control of growth of breed cattle at Dihessé breeding farm in Congo Brazaville. Pak. J. Nutr., 9 (5): 475-480, doi: 10.3923/ pjn.2010.475.480 Beeler S., 2006. Conflits entre agriculteurs et eleveurs au nord-ouest du Mali, dossier n° 141. IIED, Londres, UK Boujenane I., Aissa H., 2008. Reproductive and milk performances of Holstein and Montbeliarde cows in Morocco. Rev. Elev. Med. Vet. Pays Trop., 61 (3-4): 191-196, doi: 10.19182/remvt.9988 Coghlan A., 2011. A little book of R for Bioinformatics. Release 0.1. Wellcome Trust Sanger Institute, Cambridge, UK Denbarga Y., Woldegebriel B., Sheferaw D., 2012. Reproductive performance of Boran cows at Tates, a cattle breeding center. Adv. Bio. Res., 6 (3): 101105 Kashoma I.P.B., Luziga C., Werema C.W., Shirima G.A., Ndossi D., 2011. Predicting body weight of Tanzania shorthorn zebu cattle using heart girth measurements. Livest. Res. Rural Dev., 23: e94 Kibwana D.K., Makumyaviri A.M., Hornick J.L., 2012. Extensive farming practices and cattle performances of the local breed or crossed with exotic dairy breeds in the Democratic Republic of Congo. Rev. Elev. Med. Vet. Pays Trop., 65 (3-4): 67-74, doi: doi.org/10.19182/remvt.10125 Marichatou H., Abdoulaye S.G., Kanwe A.B., 2005. Production laitière de la race Gudhali et croissance des jeunes purs et croisés, en zone périurbaine de Bobo-Dioulasso (Burkina Faso). Cah. Agric., 14 (3) : 291-296 Martin P., 1967. Une application des fonctions de Gompertz à l’étude de la fécondité d’une cohorte. Population, 22 (6) : 1085-1096, www. persee.fr/web/revues/home/prescript/article/pop_0032-4663_1967_ num_22_6_11238 Nicholson M.J., Sayers A.R., 1987. Relatioships between body weight, condition score and heart girth changes in Boran cattle. Trop. Anim. Health Prod., 19: 115-120, doi: 10.1007/BF02297329 Poivey J.P., Landais E., Seitz J.L., 1980. Use of body measurements to estimate liveweigbt in local taurine breeds of Ivory Coast. Rev. Elev. Med. Vet. Pays Trop., 33 (3): 311-317, doi: doi.org/10.19182/remvt.8217 Denis J.P., 1971. Interval between calvings in Gobra zebu cattle (Senegalese Peulh). Rev. Elev. Med. Vet. Pays Trop., 24 (4): 635-647, doi: 10.19182/ remvt.7725 Porter T., Kebread E., Darmani K.H., Lopez S., Strathe A.B., France J., 2010. Flexible alternatives to the Gompertz equation for describing growth with age in turkey hens. Poult. Sci., 89 (2): 371-378, doi: 10.3382/ps.200900141 Dibanzilua M.N.M., Tandu S.F., Ngomo L.A., Compere R., 1995. Performances pondérales des veaux issus de trois races locales de l’Ituri (Haut-Zaïre). Tropicultura, 13 (2) : 74-78 Sokouri D.P., Yapi-Gnaore C.V., N’guetta A.S.P., Loukou N.E., Kouao B.J., Toure G., Kouassi A., Sangare A., 2010. Performances de reproduction des races bovines locales de Côte d’Ivoire. J. Appl. Biosci., 36 : 2353- 2359 Dodo K., Pandey V.S., Illiassou M.S., 2001. Weight estimation from body measurements of the Azawak zebu in Niger. Rev. Elev. Med. Vet. Pays Trop., 54 (1): 63-68, doi: 10.19182/remvt.9808 Symoens C., Hounsou-Ve G., 1991. Barymetric data in Borgou cattle breed in Northeast Benin. Rev. Elev. Med. vet. Pays trop., 44 (4): 487-490, doi: 10.19182/remvt.9158 France J., Dijkstra J., Dhanoa M.S., 1996. Growth functions and their application in animal sciences. Ann. Zootech., 45: 165-174, doi: 10.1051/ animres:19960637 Turner M.D., Mcpeak J.G., Ayantunde A., 2014. The role of livestock mobility in the livelihood strategies of rural peoples in semi-arid West Africa. Hum. Ecol., 42 (2): 231-247, doi: 10.1007/s10745-013-9636-2 Habib M.A., Bhuiyan A.K.F., Amin M.R., 2010. Reproductive performance of Red Chittagong cattle in a nucleus herd. Bangladesh J. Anim. Sci., 39. www.banglajol.info/index.php/BJAS/article/view/9673/71 Youssao A.K.I., Ahissou A., Toure Z., Leroy P.L., 2000. Productivity of the Borgu breed at the Okpara breeding farm in Benin. Rev. Elev. Med. Vet. Pays Trop., 53 (1): 67-74, doi: 10.19182/remvt.9766 Revue d’élevage et de médecine vétérinaire des pays tropicaux, 2017, 70 (4) : 115-120 Achard F., Chanono M., 1995. Un système d’élevage performant bien adapté à l’aridité à Toukounous, dans le sahel Nigérien. Sécheresse, 2 (6) : 215222 119 ■ PRODUCTIONS ANIMALES ET PRODUITS ANIMAUX Zootechnical characterization of the Azawak zebu breed in Mali Summary Resumen Touré A., Antoine-Moussiaux N., Kouriba A., Leroy P., Moula N. Zootechnical characterization and barymetric formula of the Azawak zebu breed in Menaka, Northern Mali Touré A., Antoine-Moussiaux N., Kouriba A., Leroy P., Moula N. Caracterización zootécnica y fórmula barimétrica de la raza Cebú Azawak en Menaka, Norte de Mali The objective of this study was to contribute to the preservation and improvement of the performance of the Azawak zebu in Mali by proposing an estimation of the growth and reproduction performances of this cattle breed reared in the north of the country. Monitoring involved 1129 animals in four townships in Menaka Circle, between November 2009 and February 2010. At 30 days, males weighed on average 39 ± 10 kg and females 32 ± 4 kg. No significant differences (p > 0.05) were recorded between the adult weights of bulls (370 ± 88 kg) and cows (327 ± 46 kg). The age at first calving was 50 ± 11 months with a calving interval of 16 ± 4 months. The correlations of weight with the thoracic girdle (r = 0.95) and the height at the withers (r = 0.94) were the highest. Barymetric equations were developed for each animal category (male calf / female calf, heifer / young bull, cow/bull) using polynomial weight regressions (y) on the thoracic girdle (x). These could enable livestock agents to establish a conversion table of the thoracic girdle into live weight or a barymetric measuring tape to facilitate husbandry and sanitary monitoring of animals in the field. El objetivo del presente estudio fue contribuir a la preservación y mejoramiento del rendimiento del cebú Azawak en Mali, mediante la propuesta de una estimación del rendimiento del crecimiento y la reproducción de esta raza de ganado, criado en el norte del país. El monitoreo incluyó 1129 animales en cuatro pueblos en el Círculo de Menaka, entre noviembre 2009 y febrero 2010. A los 30 días, los machos pesaron en promedio 39 ± 10 kg y las hembras 32 ± 4 kg. No se registraron diferencias significativas (p > 0,05) entre los pesos adultos de los toros (370 ± 88 kg) y las vacas (327 ± 46 kg). La edad al primer parto fue 50 ± 11 meses con un intervalo entre partos de 16 ± 4 meses. Las correlaciones entre el peso y la faja torácica (r = 0,95) y la altura a la cruz (r = 0,94) fueron las más elevadas. Se desarrollaron ecuaciones barimétricas para cada categoría animal (ternero/ternera, novilla/torete, vaca/ toro) utilizando regresiones de peso poli nominales (y) para la faja torácica (x). Estas pueden ayudar a los agentes ganaderos a establecer una tabla de conversión de la faja torácica en peso vivo o una cinta de medida barimétrica para facilitar el seguimiento de cría y sanitario de los animales en el campo. Revue d’élevage et de médecine vétérinaire des pays tropicaux, 2017, 70 (4) : 115-120 Keywords: Bos indicus, Azawak cattle, growth, reproductive performance, body weight, Mali 120 Palabras clave: Bos indicus, ganado bovino Azawak, crecimiento, reproductividad, peso corporal, Malí
https://openalex.org/W3158141643	https://www.degruyter.com/document/doi/10.1515/jmbm-2021-0003/pdf	English	null	Design and optimization of differential capacitive micro accelerometer for vibration measurement	Journal of mechanical behaviour of materials/Journal of the mechanical behavior of materials	2,021	cc-by	4,905	Research Article Kumar Gomathi, Arunachalam Balaji, and Thangaraj Mrunalini Design and optimization of differential capacitive micro accelerometer for vibration measurement ity [1]. A Capacitive type accelerometer is used instead of piezoelectric to improve the sensitivity. Capacitive micro- accelerometer has the advantage of low drift, low power consumption, low-temperature dependence, minimal cost and high sensitivity [2]. MEMS accelerometer becomes more popular and finds a place in automotive and precision ap- plications [3]. A high dynamic range accelerometer (g>50) finds its applications in the automotive sector (airbag de- ployment), biodynamic measurement, flutter testing, and high shock measurement, While a low-g accelerometer is used in large volume products like tablets, remote control system, smartphones, gaming system and toys [4]. A dif- ferential capacitive micro accelerometer is used to mea- sure the hand-arm vibration in different brick structures [5]. A bulk micromachined in-plane capacitive accelerometer fabricated with an improved process flow, by etching only one-fifth of the wafer thickness at the back of silicon while forming the bar-structure electrode [6]. It greatly lowers the footing effect during deep reactive ion etching (DRIE), and increases the proof mass by 54% compared to the tra- ditional way, resulting in both improved device quality and a higher yield rate [7]. https://doi.org/10.1515/jmbm-2021-0003 Received Jun 28, 2020; accepted Mar 01, 2021 https://doi.org/10.1515/jmbm-2021-0003 Received Jun 28, 2020; accepted Mar 01, 2021 Abstract: This paper deals with the design and optimization of a differential capacitive micro accelerometer for better displacement since other types of micro accelerometer lags in sensitivity and linearity. To overcome this problem, a capacitive area-changed technique is adopted to improve the sensitivity even in a wide acceleration range (0-100 g). The linearity is improved by designing a U-folded suspen- sion. The movable mass of the accelerometer is designed with many fingers connected in parallel and suspended over the stationary electrodes. This arrangement gives the differential comb-type capacitive accelerometer. The area changed capacitive accelerometer is designed using In- tellisuite 8.6 Software. Design parameters such as spring width and radius, length, and width of the proof mass are optimized using Minitab 17 software. Mechanical sensitivity of 0.3506 µm/g and Electrical sensitivity of 4.706 µF/g are achieved. The highest displacement of 7.899 µm is obtained with a cross-axis sensitivity of 0.47%. Keywords: MEMS, capacitive accelerometer, U-folded sus- pension, Minitab 17 software, Intellisuite 8.6 software An Area-changed accelerometer uses a ribbed finger structure on the movable mass as a differential capacitor and it is suspended over stationary electrodes. 1 Introduction The technology from the last two decades introduced Micro Electro Mechanical Systems (MEMS) in many fields. MEMS accelerometers are used to sense 1-axis, 2-axis, or 3-axis ac- celeration. The acceleration is measured by an accelerome- ter using capacitive, piezoelectric, piezoresistive, optical, tunneling, and resonance transductions. Piezoelectric and piezoresistive accelerometer lags in sensitivity and linear- Corresponding Author: Kumar Gomathi: Mechatronics Engineer- ing, Kongu Engineering College, Perundurai, Tamilnadu, 638060, India; Email: gomathik@kongu.ac.in Arunachalam Balaji: Mechatronics Engineering, Kongu Engineer- ing College, Perundurai, Tamilnadu, 638060, India Thangaraj Mrunalini: Electronics and Instrumentation Engineer- ing, Kongu Engineering College, Perundurai, Tamilnadu, 638060, India Corresponding Author: Kumar Gomathi: Mechatronics Engineer- ing, Kongu Engineering College, Perundurai, Tamilnadu, 638060, India; Email: gomathik@kongu.ac.in Research Article Kumar Gomathi, Arunachalam Balaji, and Thangaraj Mrunalini Design and optimization of differential capacitive micro accelerometer for vibration measurement The differen- tial capacitance is varied by varying the overlapping area of the proof mass and the fixed electrodes. High sensitivity can be obtained either by increasing the proof mass or lowering the spring constant of the suspension beam. Mechanical sensitivity of an area-changed capacitive accelerometer can be enhanced by optimizing the device geometry. Maximum displacement can be achieved by selecting the proper de- sign structure of the area changed capacitive accelerometer. Simulation of the accelerometer can be done using MEMS CAD tools such as Intellisuite 8.6, Coventorware 2001, and Ansys software [8, 9]. This work is licensed under the Creative Commons Attribution Journal of the Mechanical Behavior of Materials 2021; 30:19–27 eter is excited along the sense direction with acceleration ‘a’, the proof mass ‘m’, suspended by a beam or spring is displaced under the effect of the inertial force in a direction opposite to the applied acceleration.f To obtain the force equation of the proposed system, Newton’s law of motion is used. The free-body diagram of the proposed accelerometer model and the free body diagram are shownin Figures 2 and 3. Total force (Fa) acting on the system is given by New- ton’s second law of motion, which states that force devel- oped by a body is directly proportional to the product of the mass of the body and its acceleration. To apply the same model for differential capacitor area changed type, it needs some modification to withstand a high dynamic range and also possess better sensitivity and linearity. To improve the dynamic range, four suspension springs are introduced with two dashpots. Hence the total force will get divided equally into all four springs. Fa = ma (1) (1) Fa = Fm + FK1 + FK2 + FK3 + FK4 + FB1 + FB2 (2) (2) Figure 1: Dynamic model of basic accelerometer Where: M – Proof mass (kg), B1-B2 – Dashpot, K1-K2-K3-K4 – Spring constant (N/m) Figure 2: Dynamic model of the proposed design Where: Fa – applied force, Fm – inertial force of proof mass, FK1, FK2, FK3, FK4 – spring force, FB1, FB2 – Damping force. F E (2) Fm – inertial force of proof mass, FK1, FK2, FK3, FK4 – spring force, FB1, FB2 – Damping force. FK1, FK2, FK3, FK4 – spring force, FB1, FB2 – Damping force. From Eq. (2) Fa = M d2x dt2 + K1x + K2x + K3x + K4 + B1 dx dt (3) + B2 dx dt (3) Figure 1: Dynamic model of basic accelerometer Figure 1: Dynamic model of basic accelerometer Taking Laplace Transformation in Eq. (3), Taking Laplace Transformation in Eq. (3), Fa = MS2X(S) + K1X(S) + K2X(S) + K3X(S) (4) + K4X(S) + B1SX(S) + B2SX(S) Fa = (MS2 + K1 + K2 + K3 + K4 + B1S + B2S)X(S) X(S)/Fa = 1/(MS2 + K1 + K2 + K3 + K4 + B1S + B2S)X(S) (4) Where: The above derived Eq. (4) states the final mathematical modelling of the proposed accelerometer design for high- g application. 2 Mass-spring-damper system Arunachalam Balaji: Mechatronics Engineering, Kongu Engineer- ing College, Perundurai, Tamilnadu, 638060, India An accelerometer is dynamically modeled as shown in Fi- gure 1 a simple spring-mass damper system. The accelerom- Thangaraj Mrunalini: Electronics and Instrumentation Engineer- ing, Kongu Engineering College, Perundurai, Tamilnadu, 638060, India \| K. Gomathi et al. 20 With the required specification for high-g system, the dimension and mass of the each components of the system can be calculated. Figure 2: Dynamic model of the proposed design Figure 3: Free body diagram Proof mass, m Proof mass, m m = ρsi(WpLpTp + NWFLFt) (5) (5) Where ρSi is the density of silicon, wp – width of the proof mass, Lp and Tp are length and thickness of proof mass, N is the number of sensing movable fingers, WF, LF, t are width, length and thickness of sensing fingers respectively. i Spring constant, Kx kx = (6) NE(2L1 + πR1)W2 1T1 2(2L4 1 + 4πR1L2 1 + 24R2 1L2 1 + 6πR2 1L1 + 3π2R4 1 −24R4 1) (N/M) (6) Figure 3: Free body diagram Figure 3: Free body diagram Where, E = young’s modulus, L1 & L2 length of the folded beam, R1 and W1 are the radii of U-curvature of spring Design and optimization of micro accelerometer for vibration m Design and optimization of m Design and optimization of micro accelerometer for vibration measurement \| 21 and width of spring, T1 is the thickness of spring, N is the number of folded U-spring. 3 Design using Intellisuite 8.6 Natural frequency, fx︁ Natural frequency, fx 3.1 Mask layer design fx = wx 2π = √︁ Kx m 2π (7) e o s h Micro Accelerometers are designed and fabricated using various modules available in Intellisuite 8.6 software. Intel- liMask is a mask layer editor from Intellisuite 8.6 software, it provides the environment to create, edit and modify mask design’s for MEMS devices From the mathematical model- ing results, the design parameters for differential capacitive area changed micro accelerometer has been obtained and tabulated in Table 1. The bottom electrode and variable top electrode of the area changed micro accelerometer structure appeared to be a differential capacitance arrangement. Capacitance is measured while varying the area of an accelerometer which is given in Eqs (3)–(13). Capacitance, C = ε0εr A d (8) C = ε0εr WL d (9) C = ε0εr A d (8) C = ε0εr WL d (9) (8) C ε0εr d (8) C = ε0εr WL d (9) When the area of the accelerometer is changed then the capacitance equation is rewritten as C = N εLF(WF −Wd) 2d (10) Where: N = Number of proof mass fingers WF = width of proof mass fingers Wd = gap between the fixed electrodes d = distance between fixed and movable electrode Table 1: Dimensions for parameter design S.NO Parameters Dimension 1 Proof mass Length 300 µm Width 285 µm Thickness 40 µm 2 Suspension spring Length 285 µm Width 60 µm Thickness 40 µm 3 Material Silicon <110> When the area of the accelerometer is changed then the capacitance equation is rewritten as C = N εLF(WF −Wd) 2d (10) Where: e e: N = Number of proof mass fingersi 3 M t i l i WF = width of proof mass fingers i Wd = gap between the fixed electrodesi i d = distance between fixed and movable electrodei i d = distance between fixed and movable electrodei i The capacitance is amplified when the area of the ca- pacitor is moved in a forward direction and capacitance is decreasing when the area of the capacitor is moved in quash direction. The suspension beams are placed outside the plane of the micro accelerometer (proof mass). Three mask layers were modeled and developed in IntelliMask and shown in Figure 4. 3.2 Fabrication of micro accelerometer The fabrication process flow sequence can be viewed using the inbuilt animation module FabViewer in IntelliFab. This sequence can also be saved in an image sequence, which is shown in Figure 5. Initially, a Silicon substrate with an orientation of 110 is taken as a base material. The mask The above-obtained displacement at the X axis is not appropriate for general application. So the design is fur- ther processed for optimization to produce better displace- ment (Eq. 3). Figure 5: Fabrication process flow sequence for design Figure 5: Fabrication process flow sequence for design 5 Electrode design Electrodes are used to measure the acc ing sudden mechanical excitation or v type accelerometers are widely used in because of their high sensitivity and lo ficient of expansion. Among different c techniques, the differential capacitance more attention among researchers due formance than the normal capacitance The basic capacitance formula for m tion data is, C = εoεr (︂A d )︂ Where: C – capacitance between plates εr – relative permittivity εo – permittivity of air A – Area of electrode plates d – Distance between electrode plates Figure 7 shows the difference betw pacitance and differential capacitance For differential capacitance, the genera 4 Design analysis The fabrication files are then exported to the Thermo Electro Mechanical analysis (TEM) module of Intellisuite 8.6. This allows carrying out static, dynamic and frequency analysis over the fabricated designs. A load of 100 g is applied on the x-axis, 1g on the y-axis and 1g on the z-axis and the mesh analysis is done. The displacement for the given load and the displacement of the cross axis is shown in Figure 6. The above-obtained displacement at the X axis is not appropriate for general application. So the design is fur- ther processed for optimization to produce better displace- ment (Eq. 3). The fabrication files are then exported to the Thermo Electro Mechanical analysis (TEM) module of Intellisuite 8.6. This allows carrying out static, dynamic and frequency analysis over the fabricated designs. A load of 100 g is applied on the x-axis, 1g on the y-axis and 1g on the z-axis and the mesh analysis is done. The displacement for the given load and the displacement of the cross axis is shown in Figure 6. 22 \| K. Gomathi et al. 22 \| K. Gomathi et al. 22 \| K. Gomathi et al. 22 layer is created with a U-shaped spring. The deep reactive Ion Etching is done after the mask layer is created. Similarly, a fixed mask layer 1 is created. Then differential capacitance arrangement is fabricated after etching. Finally, the top and bottom view of the fabricated accelerometer is shown. The proof mass is deposited over the fixed electrode through the sacrificial insulating layer. Capacitance is measured between the top and bottom electrodes after removing the sacrificial layer. In Figure 4 U-shaped spring has been placed instead of the folded beam to improve linearity. In the design layer, 0 lengths and width of the U-spring have been chosen as 285 µm × 60 µm. Another design layer 2 for trenches forma- tion in proof mass with the same length and width has been varied to 285 µm × 32.5 µm. Finally, the layer 3 fixed elec- trode differential capacitor plate is designed with length and breadth as 275 µm × 22.5 µm. All this layer will have their effect in improving the displacement of proof mass furthermore when the micro accelerometer operating at its full range [10]. Anchors are placed in-plane with the proof mass and suspension springs are placed between these anchors. This design has been expected to provide high linearity and very low cross-axis sensitivity. All the above-created models are used for the fabrication process. 3.1 Mask layer design Mechanical sensitivity of the area changed Capacitive accelerometer is expressed as Figure 4: Mask layout for design parameter ) r, e- d ) ) Figure 4: Mask layout for design parameter Sc = 2ε0Nf h(Lf −∆Lf)Sd d2 0 (11) Where ∆Lf is the length not covered of the mobile finger, and if it is considered that much ∆Lf lower (∆Lf ∼0). Me- chanical sensitivity of the accelerometer is finally derived as S = 2εNLF D (12) where: ε = Permittivity of air N = Number of fingers LF = Length of the fingers (µm) D = Distance between the top electrode (µm) Cross axis sensitivity is measured as Cross axis sensitivity = (13) Displacement at Y axis/Displacement at X axis Figure 4: Mask layout for design parameter Sc = 2ε0Nf h(Lf −∆Lf)Sd d2 0 (11) (11) Where ∆Lf is the length not covered of the mobile finger, and if it is considered that much ∆Lf lower (∆Lf ∼0). Me- chanical sensitivity of the accelerometer is finally derived S = 2εNLF D (12) (12) where: where: ε = Permittivity of air N = Number of fingers LF = Length of the fingers (µm) D = Distance between the top electrode (µm) Cross axis sensitivity is measured as Cross axis sensitivity = (13) Displacement at Y axis/Displacement at X axis Figure 4: Mask layout for design parameter (13) Figure 4: Mask layout for design parameter Design and optimization of micro accelerometer for vibration measurement Design and optimization of micro accelerometer for vibration measurement 23 Figure 6: Displacement at axis x, y, z Figure 6: Displacement at axis x, y, z Figure 7: Differences between ordinary capacitance and differential capacitance 5 Electrode design Electrodes are used to measure the acceleration data dur- ing sudden mechanical excitation or vibration. Capacitive type accelerometers are widely used in most applications, because of their high sensitivity and low-temperature coef- ficient of expansion. Among different capacitance sensing techniques, the differential capacitance technique acquires more attention among researchers due to its improved per- formance than the normal capacitance method. The basic capacitance formula for measuring accelera- tion data is, C = εoεr (︂A d )︂ (14) (14) C – capacitance between plates Figure 7 shows the difference between the regular ca- pacitance and differential capacitance sensing techniques. For differential capacitance, the general formula is written Figure 5: Fabrication process flow sequence for design Design and optimization of micro accelerometer for vibration measurement \| 23 Figure 6: Displacement at axis x, y, z Figure 7: Differences between ordinary capacitance and differential capacitance to be, C = εoεr {︂l([b + ∆x] −[b −∆x]) d }︂ (15) Where: C – capacitance between plates εr – relative permittivity εo – permittivity of air l – Length of overlapping area of electrode b – Breadth of overlapping area of electrode ∆x – change in overlapping length of electrode d – Distance between electrode plates 6 Optimization of micro-accelerometer 6.1 Optimisation techniques Three main types of problems can be handled by the opti- mization techniques are single variable functions, multi- variable functions with no constraints, multivariable func- tions with both equality and inequality constraints. In problems with equality constraints, the Lagrange multi- plier method can be used whereas the Kuhn-Tucker condi- tions can be used to identify the optimum solution for the problems with inequality constraints. In this present work, Taguchi Method is used where the design of experiments is carried out for the optimization process (Eq. 15). Various designs are modeled using Intellisuite 8.6 is shown in Figure 8 and the resultant displacement at the x, the y axis is measured and cross-axis sensitivity is calcu- lated by using Eq. (12). In Table 2 correlating all the designs, Design 5 shows maximum displacement at X-axis and least cross-axis sensitivity when compared to all designs. Hence the optimized design (design No. 5) is further modified and Design and optimization of micro accelerometer for vibration measurement \| 23 Figure 6: Displacement at axis x, y, z Design and optimization of micro accelerometer for vibration measurement 25 Table 2: Design parameter and their result Design No. Spring width (µm) Mass length (µm) Mass width (µm) Spring arc radius (µm) Displacement (µm) Cross axis sensitivity (%) X Y 1 5 300 285 25 0.0013152 0.000194 14.75281326 2 5 1400 1242.5 37.5 0.0987405 0.0066004 6.684551932 3 5 2500 2200 75 0.306031 0.10393 33.96061183 4 12.5 300 1242.5 75 0.143323 0.0062926 4.39053048 5 12.5 1400 2200 25 7.89953 0.0455015 0.576002623 6 12.5 2500 285 37.5 0.0023013 0.0004069 17.68035145 7 20 300 2200 37.5 0.694688 0.0052972 0.762527926 8 20 1400 285 75 0.0009006 0.0002855 31.70273722 9 20 2500 1242.5 25 0.0024304 0.000181 7.448536643 ments. The displacement data for applied input accelera- tion was shown in Figure 9. mass width are 285, 1242.5 and 2200 µm and the spring arc radius are 25, 37.5 and 75 µm respectively. Next, the spring width was maintained constant as 12.5 µm for design 4, 5 & 6 and 20 µm for design 7, 8 & 9, and the mass length, mass width and spring arc radius are maintained as maintained in design 1, 2 & 3 in the order of design 3, 2 & 1. From all these designs, comparatively, design 5 shows the least cross-axis sensitivity (%). From Figure 9, the displacement for 100 g of accelera- tion applied on micro accelerometer was measured to be 9.848 µm at X-axis whereas the displacement of the ac- celerometer which does not contain holes on its proof mass is recorded as 7.899 µm. It is due to the holes in proof mass that increase the displacement of the accelerometer. Also, it helps in improving the sensitivity of the device. The hole on the proof mass will not cause any damping issues. The mass of the optimized design is 3.619e−7 kg, and the spring constant, k is 6.2642 N/m. 6.1 Optimisation techniques Three main types of problems can be handled by the opti- mization techniques are single variable functions, multi- variable functions with no constraints, multivariable func- tions with both equality and inequality constraints. In problems with equality constraints, the Lagrange multi- plier method can be used whereas the Kuhn-Tucker condi- tions can be used to identify the optimum solution for the problems with inequality constraints. In this present work, Taguchi Method is used where the design of experiments is carried out for the optimization process (Eq. 15). Figure 7: Differences between ordinary capacitance and differential capacitance C = εoεr {︂l([b + ∆x] −[b −∆x]) d }︂ (15) Where: C – capacitance between plates εr – relative permittivity εo – permittivity of air l – Length of overlapping area of electrode b – Breadth of overlapping area of electrode ∆x – change in overlapping length of electrode d – Distance between electrode plates C – capacitance between plates C – capacitance between plates εr – relative permittivity εo – permittivity of air l – Length of overlapping area of electrode b – Breadth of overlapping area of electrode ∆x – change in overlapping length of electrode d – Distance between electrode plates Various designs are modeled using Intellisuite 8.6 is shown in Figure 8 and the resultant displacement at the x, the y axis is measured and cross-axis sensitivity is calcu- lated by using Eq. (12). In Table 2 correlating all the designs, Design 5 shows maximum displacement at X-axis and least cross-axis sensitivity when compared to all designs. Hence the optimized design (design No. 5) is further modified and performance is analyzed. The spring width was maintained constant as 5 µm for the first three designs and the mass length, mass width and spring arc radius are varied. For the first, second and third design the mass length is 300, 1400 and 2500 µm, the 24 \| K. Gomathi et al. Figure 8: Various design modeled Figure 8: Various design modeled Figure 8: Various design modeled Design and optimization of micro accelerometer for vibration measurement 7 Static analysis The occurrence of cross-axis sensitivity for the acceler- ation originated out of the sensing axis has to be analyzed. This will avoid the further occurrence of an error in the out- Based on analysis results, the performance of the designed micro accelerometer was tested with the system require- Figure 9: Optimised design displacement at 100 g Figure 9: Optimised design displacement at 100 g 26 \| K. Gomathi et al. 26 Table 3: Optimised design result Acceleration (g) Displacement (µm) Cross axis sensitivity (%) X Y 10 0.984828 0.00570218 0.579002628 20 1.96965 0.0102272 0.519239459 30 2.95447 0.0147522 0.499317983 40 3.93929 0.019272 0.489225216 50 4.92411 0.0238022 0.483380753 60 5.90893 0.0283272 0.479396439 70 6.89376 0.0328523 0.476551258 80 7.87858 0.0373773 0.47441671 90 8.8634 0.0419023 0.472756504 100 9.84822 0.0464273 0.471428339 Table 4: Output parameters of designed micro accelerometer Parameters Achieved Operating range 0-100 g Natural frequency (Hz) 662.15 Cross axis sensitivity (%) 0.47 Mechanical sensitivity (µm/g) 0.3506 Electrical sensitivity (µF/g) 4.706 Material used Silicon <110> Sensing technique The area changed differential capacitor technique Figure 10: Cross axis sensitivity vs. acceleration Figure 11: Displacement vs. acceleration Table 3: Optimised design result Acceleration (g) Displacement (µm) Cross axis sensitivity (%) X Y 10 0.984828 0.00570218 0.579002628 20 1.96965 0.0102272 0.519239459 30 2.95447 0.0147522 0.499317983 40 3.93929 0.019272 0.489225216 50 4.92411 0.0238022 0.483380753 60 5.90893 0.0283272 0.479396439 70 6.89376 0.0328523 0.476551258 80 7.87858 0.0373773 0.47441671 90 8.8634 0.0419023 0.472756504 100 9.84822 0.0464273 0.471428339 Acceleration (g) Displacement (µm) Cross axis sensitivity (%) X Y 10 0.984828 0.00570218 0.579002628 20 1.96965 0.0102272 0.519239459 30 2.95447 0.0147522 0.499317983 40 3.93929 0.019272 0.489225216 50 4.92411 0.0238022 0.483380753 60 5.90893 0.0283272 0.479396439 70 6.89376 0.0328523 0.476551258 80 7.87858 0.0373773 0.47441671 90 8.8634 0.0419023 0.472756504 100 9.84822 0.0464273 0.471428339 Table 4: Output parameters of designed micro accelerometer Parameters Achieved Operating range 0-100 g Natural frequency (Hz) 662.15 Cross axis sensitivity (%) 0.47 Mechanical sensitivity (µm/g) 0.3506 Electrical sensitivity (µF/g) 4.706 Material used Silicon <110> Sensing technique The area changed differential capacitor technique Table 4: Output parameters of designed micro accelerometer Table 4: Output parameters of designed micro accelerometer Parameters Achieved Operating range 0-100 g Natural frequency (Hz) 662.15 Cross axis sensitivity (%) 0.47 Mechanical sensitivity (µm/g) 0.3506 Electrical sensitivity (µF/g) 4.706 Material used Silicon <110> Sensing technique The area changed differential capacitor technique Figure 10: Cross axis sensitivity vs. acceleration Figure 11: Displacement vs. 8 Conclusions f [3] Li C, Xue Y, Jia P, Jia M, Sun B, Xiong JJJoS. A Wireless Passive Vibration Sensor Based on High-Temperature Ceramic for Harsh Environment. 2021;2021. f [3] Li C, Xue Y, Jia P, Jia M, Sun B, Xiong JJJoS. A Wireless Passive Vibration Sensor Based on High-Temperature Ceramic for Harsh Environment. 2021;2021. In the present work, an area changed differential capacitive micro accelerometer has been mathematically designed and analyzed. [4] Marinho YQ, Fruett F, Giesbrecht MJEAoAI. Application of dif- ferential evolution to multi-objective tuning of vibration spec- trum analyzers based on microelectromechanical systems. 2021;97:104071. • From the mathematical results, design simulation was done using Intellisuite 8.6 software package. [5] Larkin K, Ghommem M, Serrano M, Abdelkefi AJMT. A review on vibrating beam-based micro/nano-gyroscopes. 2021:1-25. [5] Larkin K, Ghommem M, Serrano M, Abdelkefi AJMT. A review on vibrating beam-based micro/nano-gyroscopes. 2021:1-25. • To improve the sensitivity of the micro accelerometer at high shock range area changed capacitive tech- nique has been used. Instead of using folded suspen- sion beam, a U-shaped suspension beam was used. This will improve the linearity of the micro accelerom- eter. And also holes are made in the proof mass to improve the output. [6] Domenico DD, Askes H, Aifantis EC. Capturing wave dispersion in heterogeneous and microstructured materials through a three- length-scale gradient elasticity formulation. J Mech Behav Mater. 2018;27(5-6):20182002.i [7] Unger DJ. Erratum to: Clarification of terminology used in the paper: path-dependent J-integral evaluations around an elliptical hole for large deformation theory. J Mech Behav Mater. 2016;25(5- 6):189. • The performance analysis was done using the Thermo Electro Mechanical analysis module (TEM) from Intellisuite 8.6 software. [8] Balaji A, Mithil J, Gousanal JJ, editors. Design of Dual Copter for Surveillance Applications. IOP Conference Series: Mater Sci Eng. IOP Publishing; 2021. • The result shows that the designed micro accelerom- eter will work better. It can replace the existing piezoelectric and piezoresistive micro accelerome- ter, which lacks high sensitivity and linearity at high temperatures. [9] Balaji A, Mithil J, Gousanal JJ, eds. Design and Analysis of Uni- versal Gripper for Robotics Applications. IOP Conf Series: Mater Sci Eng. IOP Publishing; 2021. [10] Balaji A, Sai RR, Gousanal JJ, Mithil J, editors. Robust Design of Automatic Sheet Fixing System for Metal Roofing. IOP Conf Series: Mater Sci Eng. IOP Publishing; 2021. [11] Shankar S, Balaji A, Pramanik AJMRE. References Hence the designed micro accelerometer satisfies all the requirements and it performs well, even at its full dy- namic range. [1] Keshavarzi M, Yavand Hasani J. Design and optimization of fully differential capacitive MEMS accelerometer based on surface micromachining. Microsyst Technol. 2019;25(4):1369–77. [1] Keshavarzi M, Yavand Hasani J. Design and optimization of fully differential capacitive MEMS accelerometer based on surface micromachining. Microsyst Technol. 2019;25(4):1369–77. [2] Li R, Mohammed Z, Rasras M, Elfadel IAM, Choi DJM. Design, mod- elling and characterization of comb drive MEMS gap-changeable differential capacitive accelerometer. 2021;169:108377. Design and optimization of micro accelerometer for vibration measurement 27 Author contributions: All authors have accepted responsi- bility for the entire content of this manuscript and approved its submission. The overall performance shows that the designed mi- cro accelerometer withstands a dynamic range up to 100 g with a natural frequency of 662.15 Hz. By using the opti- mized values of spring width, spring arc radius, proof mass length, and proof mass width, the highest displacement of 9.848 µm/g is achieved. It provides a mechanical sensitiv- ity of 0.3506 µm/g and electrical sensitivity of 4.706 µF/g. Table 4 shows the system achieved results which are calcu- lated by using the above formula.i Conflict of interest: The authors state no conflict of inter- est. 7 Static analysis acceleration Table 4: Output parameters of designed micro accelerometer Parameters Achieved Operating range 0-100 g Natural frequency (Hz) 662.15 Cross axis sensitivity (%) 0.47 Mechanical sensitivity (µm/g) 0.3506 Electrical sensitivity (µF/g) 4.706 Material used Silicon <110> Sensing technique The area changed differential capacitor technique The area changed differential capacitor technique The area changed differential capacitor technique Figure 10: Cross axis sensitivity vs. acceleration Figure 11: Displacement vs. acceleration Figure 11: Displacement vs. acceleration Figure 10: Cross axis sensitivity vs. acceleration range of 0.47% to 0.57%. So the micro accelerometer will provide better performance. put signal. Table 3 shows the displacement and cross-axis sensitivity for applied acceleration (10-100 g). Graph 10 shows the response from initial acceleration to a range up to 100 g where the cross-axis sensitivity lowers gradually. From the literature survey, it is well known that cross-axis sensitivity should be within 2% of actual sensing sensitivity. For better performance, it is much necessary to limit it to 2%. The above results show that the designed micro accelerometer exhibits cross-axis sensitivity in the The displacements for applied acceleration are shown in Figure 11. Displacement of proof mass varies the overlap- ping area of the proof mass over the differential capacitive plates. The length of the overlapping area varies concerning constant width. This will cause an increment in capacitance in one capacitor plate and a decrease in capacitance in an- other plate. 8 Conclusions Optimization of turning parameters for AlSi10Mg/SCBA/SiC hybrid metal matrix com- posite using response surface methodology. Mater Res Express. 2019;6(10):106553. Funding information: The authors state no funding in- volved. Funding information: The authors state no funding in- volved.
W4248181601.txt	https://zenodo.org/records/2010441/files/article.pdf	de		Die Oberflächenformen des Festlandes	Geografiska annaler	1,922	public-domain	2,640	Review Author(s): Hans W:son Ahlmann Review by: Hans W:son Ahlmann Source: Geografiska Annaler, Vol. 4 (1922), pp. 215-218 Published by: Wiley on behalf of Swedish Society for Anthropology and Geography Stable URL: http://www.jstor.org/stable/519751 Accessed: 27-06-2016 14:56 UTC Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://about.jstor.org/terms JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org. Wiley, Swedish Society for Anthropology and Geography are collaborating with JSTOR to digitize, preserve and extend access to Geografiska Annaler This content downloaded from 139.86.7.217 on Mon, 27 Jun 2016 14:56:48 UTC All use subject to http://about.jstor.org/terms LITTERATUR. ALFRED HETTNER: Die Oberfldchenformen des Festiandes. Verlag B. G. Teubner 250 pp. Leipzig u. Berlin 1921. The book before us, which is by Mr A. Hettner, the well-known Professor of Geography at the University of Heidelberg and editor of "Geographische Zeitschrift", is no text-book, but rather a discussion of the functions, position, method etc. of modern geomorphology. It presupposes a searching knowledge of the subject treated, and is, as the Preface says, not intended for new beginners, but is even unsuitable for them, inasmuch as theories and conceptions are criticized in a way that demands a sure power of discrimination for their appreciation. The work in question is directed against W. M. Davis and his "School", and their method is declared in the very Preface itself to be "verfehlt". Further on in the book Davis gets a dig whenever an opportunity presents itself, but it is not until towards the close that the author's bitterness gets such full play that in nearly every page of the book there are such sentences as "Davis tritt mit der ganzen Naivitit des Amerikaners in die Welt, der meint, dass die Weltgeschichte erst mit Amerika und ihm beginne", and "das Gebaude (Davis) wird einstiirzen" and so on. As is well known, Davis has won many followers in Germany, especially after he became what is called an "exchange professor" at the University of Berlin in the year 90o8. This aroused annoyance on the part of many people, because the old German school was partly thrown into the shade and also because Davis' "system", as it is called, is not considered to satisfy the full demands of strict science. It is extremely valuable that the man who is perhaps Davis' foremost opponent should now put forward his criticism in a collected form. In spite of such unbalanced expressions as have been cited above, Hettner's criticism is as much based on facts as one can expect from a bitter opponent. Nevertheless he does not altogether avoid the fault of first drawing a caricature of the person attacked and then assailing that picture. Hettner's work gains in interest by the fact that he has expanded his criticism into a general survey of geomorphology as a universal science. On the other hand, when one reads through this book, one obtains a confirmation of the general truth that it is easier to criticize than to create, to tear down than to build up. This is further confirmed by the convulsive fumblings after a new system that Passarge has created on German soil to act as a counterpoise to Davis. It is not the intention in this place to defend Davis against the criticism with which he has been favoured. That can certainly be better done by others, more especially by the "master" himself. But a number of general considerations put forward by Hettner should be mentioned, especially as many of them, in the opinion of the present reviewer, contain a great deal of truth, and should be able to be of interest, not least in Sweden itself. It is with great joy that any person interested in physical geography greets such a book as Hettner's, if only for the reason that during the last few years interest in This content downloaded from 139.86.7.217 on Mon, 27 Jun 2016 14:56:48 UTC All use subject to http://about.jstor.org/terms 216 LITTERATUR morphology has undoubtedly fallen off in favour of anthropo-geography. This phenomenon is general, not only in Europe but also in America. Probably this, like so many other things, is a consequence of the war, which destroyed so many human things that man is now eager to make the best of what remains. Without casting the faintest reflection on the promising subject of anthropo-geography, the present reviewer is bound to regret this development, for geomorphology has been abandoned at the beginning of its career long before it had attained such clearness as is required to give human geography the firm topographical foundation without which it cannot build properly. The morphological foundation is now in many cases bad, if not entirely non-existent; and problems which demand a topographical basis are taken up for treatment by persons without any training in physical geography from the simple cause that they have lacked instruction and properly qualified guidance. It is with the greatest satisfaction that we notice how Hettner proclaims the independence of geomorphology as a discipline whithin the limits of geography, and strongly emphasizes its causal nature. Without this causal element, morphology passes into topographical cartography, an auxiliary science to geography, and not an independent branch thereof. The present reviewer cannot share the view that the map is the aim of geography; it is the foundation for conclusions as to causality, correlation and general laws. The map is a method that specially belongs to geography, but it is not its be-all and end-all. One thing which has been a delicate matter for geomorphology is its relation to geology. Geography, as an independent science, largely proceeded from geology, and later, in its efforts to maintain its independence, it came to take up a hostile position towards geology and to throw over structure and tectonics. Hettner's remark that Davis takes notice of the degree to which the different kinds of rock resist denudation only so far as to be able to attain clearness with regard to morphology seems quite correct to the present reviewer. The independence of geomorphology in respect of aim and means is so clear that one can without fear take as much as one likes from geology. Thus, in the opinion of the present reviewer, no clear picture of the morphology of the Alps, is obtainable without a knowlege of their structure and tectonics. A misapprehension of the essence of the Davis' method appears in Hettner's frequent remark that Davis neglects detailed forms. Apart from the fact that, as a matter of fact, Davis is not guilty of this error, but both himself and through some of his pupils has made some of the most searching and comprehensive descriptions of detailed forms, this remark reveals a profound perversion of the true facts of the case. To the present reviewer Davis' cycles stand out as a scheme for help in analysis and deduction. It is a first skeleton, which has to be clothed with flesh and blood as our knowledge of the problems widens and deepens. If it still looks naked, that is due not to the incorrectness of the method, but to the fact that morphology as a science has not yet got very far. In the next place, to the present reviewer the development of the cycle stands out as the object of our knowledge with regard to the normal development of a certain form-group through the gradual work of the forces engaged. It would seem to be difficult to deny the justifiability of such a purely scientific object. Many of Hettner's objections in principle seem to me to fall to the ground if we conceive the cycle in the way above indicated. Of late years what is known as the knowledge of landscape (Landschaf/skunde) has been boomed as perhaps the most important object of geography. This knowledge of landscape, which is to gather into itself all the elements of geography into a single unitary picture, is This content downloaded from 139.86.7.217 on Mon, 27 Jun 2016 14:56:48 UTC All use subject to http://about.jstor.org/terms LITTERATUR 217 of course an extremely important task for geography and may be its object; but the one- sided insistence on it harbours the same danger as has been pointed out above with regard to the setting aside of geomorphology by anthropo-geography. The multitude of elements that enter into the knowledge of landscape may easily conceal the lack of a through knowledge of the individual factors. The delight with which this knowledge of landscape has been embraced, therefore, does not always, when we come to think about it more closely, contain the best reasons for the study of geography. The present reviewer has previously pointed out the defect in the Davis school through the insufficient attention given to the morphological processes themselves erosion, transport, ice-movements and so on. Our knowledge of these circumstances is extremely defective, and it seems to the present reviewer that all possible energy must be devoted to them, inasmuch as in many cases they are of fundamental consequence for a deep understanding of the nature and succession of the topographical forms. So too the present reviewer's conception of the peneplain accords with that of Hettner. It seems to me, however, as if in Davis' account of the cycle the pleneplain stands out as the final goal to which the whole process of development strives but which it seldom attains. It is above all amongst a number of Davis' followers that the unfair insistence of the peneplain is carried on which has provoked both Hettner and other people into a justifiable criticism. This reckless treatment of a thing which is so problematical and rare as the peneplain, has largely contributed to the discrediting of the entire cycle theory. The question of the peneplain is connected with the problem of the entire development of the fluvial cycle. In his criticism of this also Hettner seems to me to put forward several considerations of importance. Often, undoubtedly, there is an uncertain subjective factor in the estimate of the stage of development of a valley. In certain cases all the elements point to a certain characterization; but in many cases the fact is that the character of the rock-ground, the climatological conditions and the like, render incorrect the simple scheme in which the development of the fluvial cycle is summarized according to Davis. In regions with a powerful movement of earth (solifluction) even very young valleys are provided with more or less flat sides, while in limestone regions old valleys may have steep sides. These objections, however, seem to mne to affect the principle and the value of the cycle theory just as little as the others do, but rather affect the incompleteness of our knowledge of the cycles which still exists. Every contribution to our knowledge as to the anomalies of the cycles ought to be greeted by the partizans of the new geomorphological "school" not as indications of the inutility of the cycle, but as contributions towards its perfection. When Hettner says, "dass eine genetische Klassifikation der Talformen weniger auf das Alter als auf die verschiedene Art der Vorginge und die verschiedene Art des Gesteines begrundet werden darf", he is right only if he caricatures the primary fluvial development series in such a way as to make it hold good "in all weathers". In other case Hettner's proposition breaks down, for it would seem to be difficult to deny that a valley in one and the same kind of rock does not undergo any change of form in the course of its development. As has been mentioned above, it is not the intention in this place to refute Hettner's various remarks, which, in accordance with those that have been touched on above, have a certain degree of truth on the supposition that the cycle developments hitherto put forward are given the character of indefeasible laws instead of first attempts to summarize the main points of the knowledge so far attained. 15 Geaografiska Annaler 92ar. This content downloaded from 139.86.7.217 on Mon, 27 Jun 2016 14:56:48 UTC All use subject to http://about.jstor.org/terms 218 LITTERATUR On the other hand, one remark which seems to me to hit one of the principles in the Davis body of doctrine is the criticism of Davis' one-sided' insistence on the deductive method. The present reviewer, in his morphological investigations, in connection with the cycle conception, has not been able to follow the deductive method to the extent that Davis seems to desire, but has preferred to go the way of analysis. In the future also I shall do the same, because our knowledge of morphology is still so defective, and views are so divergent, that one cannot come to results which can count upon an acceptance by any circle of readers without some analysis and exposition of the different premises. As an example it suffices to mention the hanging tributary valleys. In the opinion of the present reviewer, such valleys are not all caused by the overdeepening of the main valley by the glacial ice, and cannot therefore be taken off hand as cri- teria of such and be mentioned without commentary in the form-complex of the glacial cycle. Thus, for instance, most of the hanging tributary valleys in Hardanger Fjord, which Davis regarded as genuine, are in the reviewer's opinion false and have nothing to do with overdeepening. The deductive way of presenting things may be an object to aim at, but as yet it is a far distant one. A deductive summary of analytic investigation, on the other hand, may be justified by the fact that, if rightly used, it gives more clearly than other methods a synthesis of the region treated. Accordingly, even after the perusal of Hettner's book, the present reviewer retains the opinion of the cycle theory that he has long entertained: Davis' body of doctrine, based on the exceptional observation of things large and small and developed with logical clearness, is a new and valuable contribution towards a continually increased knowledge of topography; the cycles that have been described, especially in "Die erklirende Beschreibung der Landformen", form no completely finished scheme applying to everything everywhere but are a first attempt towards a summary view of the natural development of forms -- a summary which must be expanded, supplemented and amended as our knowledge advances; and the same applies to the terminology. The doctrine as to the geophysical processes must be given far greater scope, and greater attention should be devoted to structure and tectonics. Analysis and induction have their proper place alongside of deduction. Many thankofferings will assuredly be made, especially in Germany, in honour of Hettner's criticism of Davis. To the present reviewer the value of his book will be not the critical character of the many wise things that are said in it, but those wise things themselves as a positive addition to our knowledge of the cycles and the author's insistence on geomorphology as the fundamental part of geography. HANS W:SON AHLMANN. NEVIN M. FENNEMAN, The Circunmference of Geography.; >>The Geographical Review,, New York, I919, pp. I68--I75. With American audacity and lucidity Fenneman has treated in a few pages the question of the scope and essence of geography, and in doing so he has dwelt on the great importance of regional synthesis or ,land-knowledge> and also pointed out the character of geography as a science of distribution. He starts from an ingeniously designed circular diagram (Fig. i), which expresses ,the fundamental conception that sciences overlap and that each one of the specialized phases of geography belongs This content downloaded from 139.86.7.217 on Mon, 27 Jun 2016 14:56:48 UTC All use subject to http://about.jstor.org/terms
https://openalex.org/W2127058624	https://repository.naturalis.nl/pub/525043/BLUM2012057002003.pdf	English	null	New species of <I>Lessingianthus</I> (<I>Asteraceae</I>, <I>Vernonieae</I>) from Brazil and Paraguay	Blumea	2,012	cc-by	2,626	1. Lessingianthus cipoensis Dematt., sp. nov. — Fig. 1, 3a–c Lessingiantho vestito simile sed capitulis latioribus et folis brevioribus dif- fert. Frutex erectus, caulibus lanatus dense foliatis. Inflorescentiae cymosa, ramis 4–5, erectis. Capitula sessilia, solitaria, late campanulata, 9–11 mm altae. — Typus: G. Hatschbach 30041 (holo MBM), Brazil, Minas Gerais, Mun. Jaboticatuba, Serra do Cipó, orla matinha de galeria, capítulos lilás, ereta, 50 cm alt., 7 Aug. 1972. Following the segregation of Lessingianthus (Robinson 1988), the only modiﬁcation has been the transference of Lessingian- thus subgen. Oligocephalus H.Rob. to the genus Chrysolaena H.Rob. (Dematteis 2007). In addition, several species still included in Vernonia have been transferred and new ones were described (Díaz-Piedrahita & Obando 2002, Dematteis 2006, 2008, Dematteis & Angulo 2010). In the present paper two new species of Lessingianthus from Brazil and Paraguay are described and illustrated. Erect shrub, 50–70 cm tall. Stems rounded, densely white- tomentose, leafy to the inflorescence, 3–5 mm diam., internodes 1–2 cm along all the stem. Leaves alternate to subopposite, sessile, dense, ascending, coriaceous, 25–35 by 14–18 mm. Leaf blades ovate to elliptical, entire to denti culate, basally subcordate, acute at the apex, smooth and laxly lanate above, veins impressed, densely lanate beneath, pinnatinervate, secondary veins 4–5, slightly arcuate. Inflo- rescence seriate-cymose, erect, branches 4–5, short, with 2–3 heads. Bracts of the inflorescence leafy, densely lanate, larger than the heads or equal. Capitula sessile. Involucre widely campanulate, 9–11 mm high, 8–10 mm diam. Phyllaries 6–7 seriate, imbricate, appressed, acuminate, densely lanate, inner phyllaries ovate-lanceolate to linear, 7–8 by 1–1.5 mm, Blumea 57, 2012: 109–113 Blumea 57, 2012: 109–113 www.ingentaconnect.com/content/nhn/b RESEARCH ARTICLE http://dx.doi.org/10.3767/000651912X653813 New species of Lessingianthus (Asteraceae, Vernonieae) from Brazil and Paraguay M. Dematteis1, M.B. Angulo1 Key words Compositae new species South America taxonomy Vernonia Abstract   Two new species of the genus Lessingianthus (Vernonieae, Asteraceae) from Brazil and Paraguay are described and illustrated. Lessingianthus cipoensis is characterized by the presence of solitary heads disposed in short branches and ovate to elliptical leaves. It has a certain resemblance to L. vestitus, which has more branched inflorescences, with long branches, and lanceolate to obovate-lanceolate leaves. Lessingianthus paraguariensis is closely related to L. asteriflorus and L. mollissimus, but it can be distinguished by the broadly elliptical leaves and the large size of the outer phyllaries. Published on  16 July 2012 Published on  16 July 2012 Published on  16 July 2012 INTRODUCTION were acetolysed according to the procedure suggested by Erdtman (1966). For light microscopy (LM) the pollen samples were mounted in glycerine-jelly on glass slides and then ex- amined with a Zeiss Axioplan microscope. Permanent slides were deposited at the Palynological Laboratory of the Universi dad Nacional del Nordeste (PAL-CTES). For scanning electron microscopy (SEM), acetolysed pollen grains were ﬁrst washed in 96 %, and then 100 % alcohol, next sputtered with gold- palladium and then observed in a JEOL 5800 LV scanning electron microscope. The terminology applied for pollen grain description in general follows Erdtman (1966) and Punt et al. (2007). The genus Lessingianthus H.Rob. (Vernonieae, Asteraceae) comprises around 120 species widely distributed in Venezuela, Colombia, Peru, Brazil, Bolivia, Paraguay, Argentina and Uru- guay (Robinson 1988). Lessingianthus was initially established to accommodate the species originally arranged under Verno- nia Schreb. sect. Lepidaploa (Cass.) DC. ser. Macrocephalae Benth. & Hook. (Bentham & Hooker 1873). Almost all the spe- cies are perennial herbs, with medium or large-sized heads and seriate-cymose inflorescences (Dematteis 2006). Lessingianthus can be distinguished from the remaining Ameri- can members of the tribe by its non-glandular anther appen- dages, lack of basal style node and quadrate raphids in the achene wall (Robinson 1999). The genus is distinguished also by the surface morphology of the pollen grains and the basic chromosome number. The pollen grains have been called type “B” by Keeley & Jones (1979) and they are tricolporate, echi- nolophate, with a discontinuous tectum, germinal furrows very long, converging at poles and lacunae disposed in a regular pattern, lacking a polar lacuna (Angulo & Dematteis 2010). The basic chromosome number of the genus is x = 16, differing from the majority of the American Vernonieae with a base of x = 17 (Dematteis 2002). To examine micro-characters, florets were obtained from her- barium specimens and softened in boiling water to which a drop of detergent was added, dissected under a stereomicroscope, mounted in Hoyer’s solution (Anderson 1954) and studied with a light microscope. You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. Materials and Methods Pollen samples were obtained by removing one or two florets from herbarium specimens of the species. The pollen grains 1 Instituto de Botánica del Nordeste (UNNE-CONICET), Casilla de Correo 209, 3400 Corrientes, Argentina; 1 Instituto de Botánica del Nordeste (UNNE-CONICET), Casilla de C 209, 3400 Corrientes, Argentina; corresponding author e-mail: mdematteis@agr.unne.edu.ar. DESCRIPTIONS 1. Lessingianthus cipoensis Dematt., sp. nov. — Fig. 1, 3a–c er, transform, or build upon this work. make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be copyright holder. Nothing in this license impairs or restricts the author’s moral rights. 2. Lessingianthus paraguariensis Dematt., sp. nov. — Fig. 2, 3d–f 2. Lessingianthus paraguariensis Dematt., sp. nov. — Fig. 2, 3d–f Lessingiantho asterifloro simile sed capitulis solitaris et phyllaris aequo lon- gibus differt. Frutex erectus, caulibus gracilibus laxe foliatis. Inflorescentiae cymosa, ramis 2–3, erectis. Capitula sessilia, solitaria, hemisphaerica, 10–13 mm altae. — Typus: A. Schinini, M. Dematteis, R. Duré & M. Quintana 35897 (holo CTES; iso G, PY), Paraguay, Amambay, 27 km N de P.J. Caballero, S22°21'05", W55°55'24", en campos cerrados, suelo arenoso, quemados de la Sierra de Amambay, semi erecta, flores violáceas, follaje discolor, envés blanquecino, 6 Mar. 2002. Erect shrub, 50–80 cm tall. Stems 2–3, striate, laxly leafy, densely tomentose, whitish, 3.5–4.5 mm diam. at the base, ba- sal internodes 10–15 cm long, middle internodes 2–3 cm long. Leaves alternate, sessile, coriaceous, discolour, the greatest 6–8 by 3–4 cm, placed on the middle portion of the stem. Leaf blades broadly elliptic, entire, acute at the apex, obtuse at the base, glabrous or scarcely lanate above, white-tomentose be- neath, pinnatinervate, secondary veins 10–15, scarcely curved. Bracts of the inflorescence absent. Inflorescence corymbiform, erect, with 2–4 heads. Capitula pedunculate, peduncles 10–15 mm long, tomentose. Involucre hemispherical, 10–12 mm high, 15–17 mm diam. Phyllaries 2–3 seriate, laxly imbricate, the outer ones linear to linear-lanceolate, acute, densely tomen- tose, 10–15 by 1–1.5 mm, inner phyllaries lanceolate, acute to acuminate, glabrous to apically tomentose, 8–10 by 1.6–2 mm. Florets purple to violet. Corollas glabrous, 13–14 mm long, lobes lanceolate, 4–4.5 mm long, papilose at apex, with small glandular and eglandular hairs. Anthers basally caudate, 3.8–4 mm long, apical appendage ovate, 0.4–0.45 mm long. Style 14–15 mm long, branches linear, 3–3.5 mm long. Cypselas Distribution — The new species is only distributed in the Brazilian state of Minas Gerais. Habitat & Ecology — In gallery forest and campo cerrado habitats. Habitat & Ecology — In gallery forest and campo cerrado habitats. Additional specimens examined. Brazil, Minas Gerais, BR 267, km 207, 4 km depois de Bom Jardim de Minas, indo para Juiz de Fora, campo de altitude ao lado da estrada, subarbusto de aproximadamente 50 cm, com flores magenta e com frutos, 2 Feb. 1998, J.H.A. Dutilh & W. Marcondes Ferreira 35 (CESJ). Note — Lessingianthus cipoensis bears a resemblance to L. vestitus (Baker) H.Rob., but can be separated by its shorter and less branched inflorescence, acuminate phyllaries, ovate leaves and leaf blades 2.5–3.5 cm long. © 2012 Nationaal Herbarium Nederland 110 Blumea – Volume 57 / 2, 2012 Fig. 1   Lessingianthus cipoensis Dematt. a. Flowering branch; b. capitulum; c, d. outer phyllaries; e, f. middle phyllaries; g, h. inner phyllaries; i. corolla sh ng anthers; j. corolla lobe (all: Hatschbach 30041, MBM). c b d a g h i j e f 4 mm 2 mm 2 mm 0.5 mm 2 cm b 2 mm 0.5 mm 2 mm f d h g e 2 cm a nsis Dematt. a. Flowering branch; b. capitulum; c, d. outer phyllaries; e, f. middle phyllaries; g, h. inner phyllaries; i. corolla show- l: Hatschbach 30041, MBM). Fig. 1   Lessingianthus cipoensis Dematt. a. Flowering branch; b. capitulum; c, d. outer phyllaries; e, f. middle phyllaries; g, h. inner phyllaries; i. corolla show- ing anthers; j. corolla lobe (all: Hatschbach 30041, MBM). M. Dematteis & M.B. Angulo: New species of Lessingianthus 111 4 mm 4 mm 2 cm 2 mm 0.5 mm c b d a g h i e f 0.5 mm 2 mm 4 mm b b 4 mm a f g d c e Fig. 2   Lessingianthus paraguariensis Dematt. a. Plant; b. capitulum; c, d. outer phyllaries; e. middle phyllary; f, g. inner phyllaries; h. corolla showing anthers; i. corolla lobe (all: Schinini, Dematteis, Duré & Quintana 35897, CTES). 112 Blumea – Volume 57 / 2, 2012 middle phyllaries ovate-lanceolate, 3–5 by 1–1.2 mm, outer ones ovate, 2–3.5 by 0.7–1 mm. Florets violet. Corollas 6–8 mm long, lobes lanceolate, 2.5–3 mm long, glandular, laxly pilose at the apex. Anthers basally calcarate, thecae 3.2–3.6 mm long, apical appendages ovate, 0.3–0.4 mm long. Style 9–10 mm long, branches linear, 1.5–1.8 mm long. Cypselas obconical, ribbed, densely sericeous pubescent, 2–2.5 mm long, with idioblasts and prismatic crystals in the wall. Pappus biseriate, greyish, inner bristles 6–7 mm long, outer scales linear-lanceolate, ﬁmbriate, 1.6–1.8 mm long. Pollen grains oblate-spheroidal (P/E = 0.99), tricolporate, echinolophate, germinal furrows very long, converging at poles. Polar diam. 59.84–(61.2)–62.56 μm, equatorial axis 59.8–(61.8)–63.9 μm. Exine, excluding the spines, 8.2–8.4 µm thickness. Lacunae 12.2–14.9 µm diam., disposed in a regular pattern, polar lacuna absent. Spines 2.5–3.9 long with apex acute. gulose above. Lessingianthus cipoensis has stems tomentose, leaves ovate to elliptical, phyllaries 6–7 seriate and leaf blades smooth on the adaxial surface. 2. Lessingianthus paraguariensis Dematt., sp. nov. — Fig. 2, 3d–f Lessingianthus ves- titus has more branched inflorescences, with long branches, acute phyllaries, lanceolate to obovate-lanceolate leaves and leaf blades 10–15 cm long. The new species is also related to L. lacunosus (Mart. ex DC.) H.Rob., which has stems lanate, lanceolate leaves, phyllaries 5–6 seriate and leaf blades ru- Fig. 3   Microcharacters and pollen grains of the new species. a–c. Lessingianthus cipoensis; a. corolla lobe apex; b. pollen grain, polar view; c. pollen grain, equatorial view; d–f. L. paraguariensis; d. idioblasts; e. crystals of the achene wall; f. corolla lobe apex (a–c: Hatschbach 30041, MBM; d–f: Schinini et al. 35897, CTES). c b d a e f a c c b b a c b e f f d e Fig. 3   Microcharacters and pollen grains of the new species. a–c. Lessingianthus cipoensis; a. corolla lobe apex; b. pollen grain, polar view; c. pollen grain, equatorial view; d–f. L. paraguariensis; d. idioblasts; e. crystals of the achene wall; f. corolla lobe apex (a–c: Hatschbach 30041, MBM; d–f: Schinini et al. 35897, CTES). M. Dematteis & M.B. Angulo: New species of Lessingianthus 113 ribbed, laxly sericeous, densely covered of idioblast, raphides quadrangular to prismatic, 3.3–3.5 mm long. Pappus white, biseriate, outer scales ﬁmbriate, irregular, 1.2–2.1 mm long, inner bristles 9–10 mm long. Pollen grains oblate-spheroidal (P/E = 0.98), 3-colporate, echinolophate. Colpi long, with apices visible in polar view. Polar diam. 47.6–(48.4)–48.9 μm, equato- rial axis 48.9–(49.3)–50.3 μm. Exine thickness 5.1–5.3 μm. Tectum discontinuous. Lacunae 10–12 µm diam., disposed in a regular pattern, polar lacuna absent. Tectum surface densely microperforate and spinose; spines 2.8 μm long, acute at apex. References Anderson LE. 1954. Hoyer’s solution as a rapid mounting medium for bryo- phytes. Bryologist 57: 242–247. Angulo MB, Dematteis M. 2010. Pollen morphology of the South American genus Lessingianthus (Vernonieae, Asteraceae) and its taxonomic implica- tions. Grana 49, 1: 12–25. Bentham G, Hooker JD. 1873. Vernonieae. Genera Plantarum. Reeve & Co., London. Dematteis M. 2002. Cytotaxonomic analysis of South American species of Vernonia (Vernonieae: Asteraceae). Botanical Journal of the Linnean Society 139: 401–408. Distribution — Paraguay (Amambay). Distribution — Paraguay (Amambay). Dematteis M. 2006. New species of Lessingianthus (Asteraceae, Vernonieae) from Central Brazil. Blumea 51: 299–304. Habitat & Ecology — In campo and on grassy savannas with dry, rocky soils. Flowering and fruiting in January and March. Dematteis M. 2007. Taxonomic notes on the genus Chrysolaena (Verno nieae, Asteraceae), including a new species endemic to Paraguay. Annales Botanici Fennici 44, 1: 56–64. Etymology — The speciﬁc epithet refers to the region where the species occur. Etymology — The speciﬁc epithet refers to the region where the species occur. Dematteis M. 2008. New species and new combinations in the South Ameri- can genus Lessingianthus (Vernonieae, Asteraceae). Edinburgh Journal of Botany 65: 359–368. Additional specimens examined. Paraguay, Amambay, in altoplanitie et declivibus Sierra de Amambay, Jan. 1907/1908, T. Rojas 10060 (BM, G). Dematteis M, Angulo MB. 2010. Additions to the genus Lessingianthus (Ver- nonieae, Asteraceae) from South America. Rodriguésia 61, 2: 233–241. Note — Lessingianthus paraguariensis is closely related to L. asteriflorus (Mart. ex DC.) H.Rob. and L. mollissimus (D.Don) H.Rob., but it can be distinguished by the broadly elliptical leaves and the size of the outer phyllaries, larger than the inner ones. Lessingianthus asteriflorus and L. mollissimus have lanceolate to oblong-lanceolate leaves and outer phyl- laries shorter than the inner ones. In addition, the new species has the larger leaves in the middle portion of the stem, while L. asteriflorus and L. mollissimus have the larger leaves at the base of the stems. Díaz-Piedrahita S, Obando S. 2002. Novedades en Vernonieae (Asteraceae) de Colombia. Revista de la Academia Colombiana de Ciencias Exactas 26: 347–351. Erdtman, G. 1966. Pollen morphology and plant taxonomy. Angiosperms. Haf- ner, New York. Keeley SC, Jones SB. 1979. Distribution of the pollen types in Vernonia (Ver- nonieae: Asteraceae). Systematic Botany 4: 195–202. Punt W, Hoen PP, Blackmore S, Nilsson S, Le Thomas A. 2007. Glossary Punt W, Hoen PP, Blackmore S, Nilsson S, Le Thomas A. 2007. Acknowledgements   We are grateful to the curators and staff of the exa mined herbaria for their collaboration and loans of specimens. Thanks to Mirtha Liliana Gómez for preparing the line drawings. This work has been supported by grants from Consejo Nacional de Investigaciones Cientíﬁcas y Tecnológicas (CONICET), Secretaría General de Ciencia y Técnica de la Universidad Nacional del Nordeste (SGCyT-UNNE) and the Myndel Botanica Foundation, which are greatly appreciated. References Glossary of pollen and spore terminology. 2nd rev. ed. LPP Foundation, Utrecht. of pollen and spore terminology. 2nd rev. ed. LPP Foundation, Utrecht. Robinson H. 1988. Studies in the Lepidaploa complex (Vernonieae: Astera- ceae). IV. The new genus Lessingianthus. Proceedings of the Biological Society of Washington 100: 929–951. Acknowledgements   We are grateful to the curators and staff of the exa mined herbaria for their collaboration and loans of specimens. Thanks to Mirtha Liliana Gómez for preparing the line drawings. This work has been supported by grants from Consejo Nacional de Investigaciones Cientíﬁcas y Tecnológicas (CONICET), Secretaría General de Ciencia y Técnica de la Universidad Nacional del Nordeste (SGCyT-UNNE) and the Myndel Botanica Foundation, which are greatly appreciated. Robinson H. 1999. Generic and subtribal classiﬁcation of American Verno nieae. Smithsonian Contributions to Botany 89: 1–116.
https://openalex.org/W3101665513	https://link.springer.com/content/pdf/10.1007%2FJHEP10%282016%29142.pdf	English	null	Kerr-Newman black holes with string corrections	The Journal of high energy physics/The journal of high energy physics	2,016	cc-by	12,029	Published for SISSA by Springer Received: June 10, 2016 Revised: October 10, 2016 Accepted: October 24, 2016 Published: October 26, 2016 Received: June 10, 2016 Revised: October 10, 2016 Accepted: October 24, 2016 Published: October 26, 2016 JHEP10(2016)142 Open Access, c⃝The Authors. Article funded by SCOAP3. B Simplifying the Lagrangian B.1 Partial gauge-ﬁxing B.2 Eliminating auxiliary ﬁelds B.3 Resulting Lagrangian Anthony M. Charles and Finn Larsen doi:10.1007/JHEP10(2016)142 doi:10.1007/JHEP10(2016)142 Contents 1 Introduction and summary 1 2 Higher-derivative N = 2 supergravity 3 2.1 Field content 3 2.2 Deﬁnitions and notation 4 2.3 Prepotential 5 2.4 Action 6 3 Minimal supergravity with (Weyl)2 corrections 7 3.1 Prepotential and Action 7 3.2 Equations of motion 8 4 Non-supersymmetric solutions 10 4.1 Einstein-Maxwell 10 4.2 Embedding 10 4.3 Simpliﬁed Lagrangian 12 5 Properties of black holes in higher-derivative gravity 14 5.1 Black hole entropy 14 5.2 OSV conjecture 15 6 Discussion 17 A Oﬀ-shell N = 2 supergravity 17 A.1 N = 2 supergravity multiplets 18 A.2 Oﬀ-shell action 18 A.3 Higher-derivative interactions 20 B Simplifying the Lagrangian 21 B.1 Partial gauge-ﬁxing 21 B.2 Eliminating auxiliary ﬁelds 21 B.3 Resulting Lagrangian 22 Contents 1 Introduction and summary 1 2 Higher-derivative N = 2 supergravity 3 2.1 Field content 3 2.2 Deﬁnitions and notation 4 2.3 Prepotential 5 2.4 Action 6 3 Minimal supergravity with (Weyl)2 corrections 7 3.1 Prepotential and Action 7 3.2 Equations of motion 8 4 Non-supersymmetric solutions 10 4.1 Einstein-Maxwell 10 4.2 Embedding 10 4.3 Simpliﬁed Lagrangian 12 5 Properties of black holes in higher-derivative gravity 14 5.1 Black hole entropy 14 5.2 OSV conjecture 15 6 Discussion 17 A Oﬀ-shell N = 2 supergravity 17 A.1 N = 2 supergravity multiplets 18 A.2 Oﬀ-shell action 18 A.3 Higher-derivative interactions 20 B Simplifying the Lagrangian 21 B.1 Partial gauge-ﬁxing 21 B.2 Eliminating auxiliary ﬁelds 21 B.3 Resulting Lagrangian 22 Contents 1 Introduction and summary 1 2 Higher-derivative N = 2 supergravity 3 2.1 Field content 3 2.2 Deﬁnitions and notation 4 2.3 Prepotential 5 2.4 Action 6 3 Minimal supergravity with (Weyl)2 corrections 7 3.1 Prepotential and Action 7 3.2 Equations of motion 8 4 Non-supersymmetric solutions 10 4.1 Einstein-Maxwell 10 4.2 Embedding 10 4.3 Simpliﬁed Lagrangian 12 5 Properties of black holes in higher-derivative gravity 14 5.1 Black hole entropy 14 5.2 OSV conjecture 15 6 Discussion 17 A Oﬀ-shell N = 2 supergravity 17 A.1 N = 2 supergravity multiplets 18 A.2 Oﬀ-shell action 18 A.3 Higher-derivative interactions 20 B Simplifying the Lagrangian 21 B.1 Partial gauge-ﬁxing 21 B.2 Eliminating auxiliary ﬁelds 21 B.3 Resulting Lagrangian 22 JHEP10(2016)142 A.3 Higher-derivative interactions Anthony M. Charles and Finn Larsen Department of Physics and Michigan Center for Theoretical Physics, University of Michigan, 450 Church Street, Ann Arbor, MI 48109-1020, U.S.A. E-mail: amchar@umich.edu, larsenf@umich.edu E-mail: amchar@umich.edu, larsenf@umich.edu Abstract: We study N = 2 supergravity with higher-derivative corrections that preserve the N = 2 supersymmetry and show that Kerr-Newman black holes are solutions to these theories. Modiﬁcations of the black hole entropy due to the higher derivatives are universal and apply even in the BPS and Schwarzschild limits. Our solutions and their entropy are greatly simpliﬁed by supersymmetry of the theory even though the black holes generally do not preserve any of the supersymmetry. Keywords: Black Holes, Black Holes in String Theory Keywords: Black Holes, Black Holes in String Theory ArXiv ePrint: 1605.07622 Open Access, c⃝The Authors. Article funded by SCOAP3. Open Access, c⃝The Authors. Article funded by SCOAP3. 1 Introduction and summary Most precision studies of black holes in string theory are carried out near the BPS limit where supersymmetry guarantees control. It is thought that various corrections become unwieldy far from this limit. Curiously, most discussions of the black hole information – 1 – paradox are carried out in the opposite limit of Schwarzschild black holes since their ge- ometries are the simplest. It is thought that this is suﬃcient to gain universal insights. Few details on the implied interpolation between the BPS and Schwarzchild limits are known. In this paper we construct families of black holes that interpolate between these limits while taking certain string corrections into account. We ﬁnd that the string corrections are surprisingly manageable. The simpliﬁcations we report are due to supersymmetry of the theories we consider. Importantly, they persist even though the black holes we construct generally do not preserve any of the supersymmetry. A convenient starting point for connection with studies that are not motivated by string theory is the 4D Einstein-Maxwell theory JHEP10(2016)142 LEM = − 1 16πGN R + 1 4FµνF µν . (1.1) (1.1) We primarily consider the standard Kerr-Newman family of solutions that includes BPS black holes and Schwarzchild black holes as special cases. A simple way to add higher-derivative terms to this theory is to consider the Gauss- Bonnet density Bonnet density LGB = αE4 = α RµνρσRµνρσ −4RµνRµν + R2 . (1.2) (1.2) This term is topological so the equations of motion are unchanged and therefore solutions remain the same. Black holes nevertheless have a diﬀerent entropy in the modiﬁed theory because the Wald entropy formula depends on the action [1–3]. Generally other linear combinations of the curvature invariants are much more com- plicated. The Weyl invariant LWeyl = γWµνρσW µνρσ = γ RµνρσRµνρσ −2RµνRµν + 1 3R2 , (1.3) (1.3) introduces the Bach tensor into the equations of motion which are then diﬃcult to analyze. In our work we are inspired by string theory and consider theories with N = 2 supersym- metry such as the supersymmetric completion of (1.3) that takes the schematic form (made precise in equation (4.17) below): LN=2 Weyl = γ1W 2 + γ2F 4 + γ3WF 2 + . . . , (1.4) (1.4) with various contractions of the tensors. 1 Introduction and summary In this case the equations of motion are even more complicated and it is not clear from the outset that it is realistic to solve them. We ﬁnd that, surprisingly, any solution to Einstein-Maxwell theory automatically solves the full theory with N = 2 supersymmetry. This will allow us to study generic non-supersymmetric solutions in the presence of higher-derivative corrections. The higher-derivative corrections modify the Wald entropy of Kerr-Newman black holes. It turns out that the combined contribution from all the terms in the supersym- metrized Weyl invariant (1.4) is precisely the same as the modiﬁcation due to the Gauss- Bonnet density (1.2) alone. In particular, the contribution from higher-derivative terms is topological. It is therefore independent of black hole parameters and can be extrapolated arbitrarily far from the BPS limit with no change. – 2 – The supersymmetrized Weyl invariant (1.4) commonly appears in low energy eﬀective actions. For example, it arises when massive string modes are integrated out. (For a discussion, see e.g. [4–6].) The terms we consider are string corrections in this sense. Our result indicates that string corrections are milder than previousely suspected. Massless modes running in virtual loops oﬀer a related quantum mechanism that gives higher-derivative terms at low energy. In previous work [7] we studied the logarithmic corrections to Kerr-Newman entropy due to such eﬀects. In general these logarithmic corrections are very complicated but upon embedding of the Kerr-Newman black hole into a theory with N ≥2 supersymmetry they greatly simplify and become independent of the black hole parameters. JHEP10(2016)142 The two classes of corrections we have considered both show that black hole entropy depends greatly on the setting. In an environment with N ≥2 supersymmetry there are considerable simpliﬁcations even for black holes that do not themselves preserve any supersymmetry. Indeed, several of the corrections to the entropy that have been analyzed precisely in the BPS limit do not depend on black hole parameters at all and so apply far oﬀextremality. This result raises hopes that the entropy of non-supersymmetric black holes can be understood precisely in a microscopic theory. This paper is organized as follows. In section 2 we present a simpliﬁed summary of oﬀ- shell N = 2 supergravity. 1 Introduction and summary (More details are given in the appendix.) In section 3 we study minimal supergravity with higher-derivative corrections in the form of a supersymmetrized Weyl invariant and derive the full equations of motion for the theory. In section 4 we embed arbitrary Einstein-Maxwell solutions into our minimal supergravity theory and show that all ﬁelds are unchanged, even for solutions that do not preserve supersymmetry. In section 5 we study properties of black holes in this embedding and ﬁnd that the correction to the black hole entropy is topological and independent of black hole parameters. Finally, in section 6 we discuss our results and potential implications for microscopic models of Kerr- Newman black holes. 2 Higher-derivative N = 2 supergravity The details of 4D oﬀ-shell N = 2 supergravity with higher-derivative interactions have been studied exhaustively [8–12]. We review some technical details in the appendix. In this section we present a more elementary and accessible discussion of N = 2 supergravity with higher-derivative corrections. 2.1 Field content We focus on the bosonic ﬁelds in N = 2 supergravity. The physical N = 2 gravity multiplet contains the metric gµν and a U(1) graviphoton ﬁeld. We further couple this theory to nV physical N = 2 vector multiplets, each comprising a U(1) gauge ﬁeld and a complex scalar. The version of the oﬀ-shell formalism we employ realizes this coupling by introducing nV + 1 vectors W I µ and nV + 1 complex scalars XI, where I = 0, . . . , nV . One of the complex scalars can be removed by symmetries and does not correspond to physical degrees of freedom. Without loss of generality, we can choose the auxiliary scalar to be X0, – 3 – Oﬀ-Shell Field Content Weyl multiplet: gµν , Aµ , T − µν , D Vector multiplets: W I µ , XI (I = 0, . . . , nV ) Physical Field Content Gravity multiplet: gµν , W 0 µ Vector multiplets: W a µ , Xa (a = 1, . . . , nV ) Auxiliary Fields X0 , Aµ , T − µν , D Table 1. Summary of the ﬁeld content in the N = 2 supergravity theory. The nV + 1 oﬀ-shell vector multiplets are indexed by I, while the nV physical vector multiplets are indexed by a. Table 1. Summary of the ﬁeld content in the N = 2 supergravity theory. The nV + 1 oﬀ-sh vector multiplets are indexed by I, while the nV physical vector multiplets are indexed by a. JHEP10(2016)142 and we will index the physical vector multiplets by a = 1, . . . , nV . The remaining gauge ﬁeld W 0 µ gets combined with the metric to form the N = 2 gravity multiplet on-shell. The complete formalism based on realization of superconformal symmetry contains many other auxiliary ﬁelds that must be carefully considered. However, for our purposes we can consistently set most of these ﬁelds to zero at the level of the action. The only ones we must retain are a U(1)R vector ﬁeld Aµ, an anti-self-dual antisymmetric tensor T − µν and a scalar D that all belong to an oﬀ-shell N = 2 Weyl multiplet with the metric. We summarize this discussion with a list of ﬁelds, from both the oﬀ-shell and the on-shell perspectives, in table 1. 2.2 Deﬁnitions and notation The only (non-composite) ﬁelds with non- zero chiral weights are the scalars XI and the anti-self-dual tensor T − µν. The ﬁelds XI and T − µν have chiral weight c = −1, while their Hermitian conjugates ¯XI and T + µν have the opposite chiral weight c = +1. The supercovariant derivative acts on these ﬁelds via where ∇µ is the ordinary covariant derivative. The only (non-composite) ﬁelds with non- zero chiral weights are the scalars XI and the anti-self-dual tensor T − µν. The ﬁelds XI and T − µν have chiral weight c = −1, while their Hermitian conjugates ¯XI and T + µν have the opposite chiral weight c = +1. The supercovariant derivative acts on these ﬁelds via DµXI = (∇µ + iAµ)XI , DµT − ρσ = (∇µ + iAµ)T − ρσ . (2.8) (2.8) The scalar operators DµDµ and ∇µ∇µ are both useful. They are distinguished by the notation □= DµDµ , ∇2 = ∇µ∇µ . (2.9) (2.9) To summarize, we present all of the ﬁelds and their corresponding chiral weight c (which determines how the supercovariant derivative (2.7) acts on the ﬁeld) in table 2. We will need to ﬁnd the equations of motion for all fundamental ﬁelds, both physical and auxiliary, but not the composite ﬁelds; those are deﬁned for notational reasons only. 2.2 Deﬁnitions and notation We will denote the ﬁeld strengths of the U(1)R gauge ﬁeld Aµ and the nV + 1 vector multiplet gauge ﬁelds W I µ as Aµν = ∂µAν −∂νAµ , F I µν = ∂µW I ν −∂νW I µ . (2.1) (2.1) The self-dual and anti-self-dual parts of these ﬁeld strengths are The self-dual and anti-self-dual parts of these ﬁeld strengths are A± µν = 1 2 Aµν ± ˜Aµν , F ±I µν = 1 2 F I µν ± ˜F I µν , (2.2) (2.2) where the dual ﬁeld strengths ˜Aµν and ˜F I µν in our conventions are ˜Aµν = −i 2εµνρσAρσ , ˜F I µν = −i 2εµνρσF ρσI . (2.3) (2.3) We denote antisymmetrized and symmetrized indices by [µν] = 1 2(µν −νµ) , (µν) = 1 2(µν + νµ) . (2.4) (2.4) – 4 – To make it manageable to present equations in the following work we deﬁne the com- posite ﬁelds F−I µν = F −I µν −1 4 ¯XIT − µν , ˆA = T − µνT −µν , ˆF − µν = −16 WµνρσT −ρσ + DT − µν + 2iAρ[µT −ρ ν] , ˆC = 32 WµνρσW µνρσ + i∗WµνρσW µνρσ + 6D2 −2AµνAµν + 2Aµν ˜Aµν −1 2T −µνDµDρT + ρν + 1 4Rµ νT − µρT +νρ + 1 256T − µνT −µνT + ρσT +ρσ , (2.5) ˆC = 32 WµνρσW µνρσ + i∗WµνρσW µνρσ + 6D2 −2AµνAµν + 2Aµν ˜Aµν −1 2T −µνDµDρT + ρν + 1 4Rµ νT − µρT +νρ + 1 256T − µνT −µνT + ρσT +ρσ , (2.5) JHEP10(2016)142 (2.5) where the dual to the Weyl tensor is where the dual to the Weyl tensor is where the dual to the Weyl tensor is ∗Wµνρσ = 1 2ε λτ µν Wρσλτ . (2.6) (2.6) The composite ﬁelds have signiﬁcance in the underlying superconformal multiplet calculus. However, in this paper we take a low-brow attitude where they represent nothing but notation for combinations of fundamental ﬁelds, both physical and auxiliary. We deﬁne the supercovariant derivative Dµ which acts on a ﬁeld φ with chiral weight c by Dµφ = (∇µ −icAµ) φ , (2.7) (2.7) where ∇µ is the ordinary covariant derivative. 2.3 Prepotential The interactions of N = 2 supergravity coupled to vector multiplets can be speciﬁed succinctly by a prepotential [13–15]. In the two-derivative theory, the prepotential is a meromorphic function of the complex scalars XI. A large class of higher-derivative cor- rections can be incorporated by considering generalized prepotentials that are functions of ˆA = T − µνT −µν as well. We will denote the prepotential by F ≡F(XI, ˆA) . (2.10) (2.10) – 5 – Fundamental Composite Field gµν W I µ XI Aµ T − µν D F− µν ˆA ˆF − µν ˆC Chiral weight 0 0 −1 0 −1 0 0 −2 −1 0 Table 2. Summary of the ﬁelds (and their corresponding chiral weight c) in our theory. The conjugate ﬁelds have opposite chiral weights. Table 2. Summary of the ﬁelds (and their corresponding chiral weight c) in our theory. The conjugate ﬁelds have opposite chiral weights. The derivatives of the prepotential are denoted The derivatives of the prepotential are denoted JHEP10(2016)142 ∂F ∂XI = FI , ∂F ∂ˆA = FA . (2.11) (2.11) The prepotential is holomorphic, so F¯I = F ¯ A = 0 (2.12) (2.12) The prepotential is homogeneous of degree two under weighted Weyl transformations where the scalar ﬁelds XI and ˆA = T − µνT −µν have Weyl weight w = 1 and w = 2, respec- tively. Thus, the prepotential satisﬁes the homogeneity relation FIXI + 2FA ˆA = 2F . (2.13) (2.13) 2.4 Action The low-energy action will then reduce to an Einstein-Hilbert action coupled to matter. 3 Minimal supergravity with (Weyl)2 corrections In this section we specialize to minimal supergravity, where gravity is coupled to a single vector ﬁeld, with higher-derivative corrections in the form of a supersymmetrized (Weyl)2 term. We will present the prepotential and action for the theory and derive the full equa- tions of motion. JHEP10(2016)142 2.4 Action We can now present the bosonic part of the N = 2 supergravity action as S = Z d4x √−g L , (2.14) (2.14) with with 8πL = −i 2(FI ¯XI −¯FIXI)R + iDµFIDµ ¯XI + h.c. + i 4FIJF−I µν F−µνJ −i 8FIF+I µν T +µν −i 32FT + µνT +µν + i 2FAIF−I µν ˆF −µν + i 2FA ˆC + i 4FAA ˆF − µν ˆF −µν + h.c. , (2.15) (2.15) where F−I µν , ˆA, ˆF − µν and ˆC are the composite ﬁelds deﬁned in (2.5), and F = F(XI, ˆA) is the prepotential discussed in section 2.3. Any solution to the equations of motion of this action must also be subject to the constraint D = −1 3R , (2.16) (2.16) which arises from making sure that the auxiliary D-ﬁeld equation of motion is consistent with the other equations of motion. The details of how we arrived at the Lagrangian (2.15) are given in the appendix. which arises from making sure that the auxiliary D-ﬁeld equation of motion is consistent with the other equations of motion. The details of how we arrived at the Lagrangian (2.15) are given in the appendix. The coeﬃcient of the Ricci scalar in the action is determined by the K¨ahler potential e−K ≡i FI ¯XI −¯FIXI . (2.17) (2.17) – 6 – – 6 – At face value this means the metric is in a non-canonical frame since the Ricci scalar normalization depends on the ﬁelds XI and ˆA. However, the theory is invariant under a local Weyl symmetry that acts as a gauge symmetry and constrains the scalars XI such that only nV of them are independent. In particular, we can gauge-ﬁx our theory and choose one of the scalars such that the K¨ahler potential is constant. The low-energy action will then reduce to an Einstein-Hilbert action coupled to matter. At face value this means the metric is in a non-canonical frame since the Ricci scalar normalization depends on the ﬁelds XI and ˆA. However, the theory is invariant under a local Weyl symmetry that acts as a gauge symmetry and constrains the scalars XI such that only nV of them are independent. In particular, we can gauge-ﬁx our theory and choose one of the scalars such that the K¨ahler potential is constant. 3.1 Prepotential and Action Following the discussion in section 2.1, the ﬁeld content for a theory with nV = 0 physical N = 2 vector multiplets is as follows. There is a Weyl multiplet containing the metric gµν and a single vector multiplet containing a physical U(1) gauge ﬁeld Wµ and a complex scalar X. The complex scalar ﬁeld will eventually be gauge-ﬁxed, leaving no physical scalars. The oﬀ-shell formalism reviewed in section 2 (and the appendix) further requires that our theory contain the auxiliary U(1)R vector ﬁeld Aµ, the auxiliary scalar D and the auxiliary antisymmetric tensor T − µν. The Lagrangian will be a function of all these ﬁelds. µ The prepotential in the minimal theory is a function only of the complex scalar X and the composite ﬁeld ˆA = T − µνT −µν. In this paper we focus on four-derivative correc- tions to minimal supergravity, which corresponds to a term in the prepotential that is linear in ˆA. Higher powers of ˆA will give rise to corrections with at least six derivatives. The homogeneity (2.13) and holomorphicity (2.12) conditions require the prepotential take the form F(X, ˆA) = −i 2X2 −c ˆA , c = c1 + ic2 ∈C . (3.1) (3.1) We can now specialize the full bosonic Lagrangian (2.15) to the minimal supergravity case deﬁned by the prepotential (3.1). Dropping all total derivative terms, we ﬁnd We can now specialize the full bosonic Lagrangian (2.15) to the minimal supergravity case deﬁned by the prepotential (3.1). Dropping all total derivative terms, we ﬁnd 8πL = −\|X\|2R + 2DµXDµ ¯X + 1 4FµνF µν −1 4Fµν XT +µν + ¯XT −µν + 1 32 X2T + µνT +µν + ¯X2T − µνT −µν + 32c2 WµνρσW µνρσ + 6D2 −2AµνAµν + 1 2(DµT −µν)(DρT + ρν) + 1 4Rµ νT − µρT +νρ + 1 512T − µνT −µνT + ρσT +ρσ . (3.2) (3.2) As we discussed in the general case, any solution is also subject to the constraint equa- tion D = −1 3R. As we discussed in the general case, any solution is also subject to the constraint equa- tion D = −1 3R. – 7 – – 7 – The coeﬃcient of the Ricci scalar is determined by the complex scalar X. As we noted in section 2.4, the local Weyl symmetry of the action allows a gauge where X is an arbitrary constant. 3.1 Prepotential and Action We will eventually assign it the conventional numerical value but for now we keep X as an independent ﬁeld. For c2 = 0 our minimal N = 2 supergravity Lagrangian (3.2) reduces to the standard two-derivative minimal supergravity, albeit presented in a somewhat unfamiliar form. The new terms are collected in the bracket preceded by the factor 32c2. They include ﬁrst of all an explicit WµνρσW µνρσ term, as we wanted, but there are many other terms as well. We interpret the entire expression proportional to c2 as the N = 2 supersymmetric completion of WµνρσW µνρσ. JHEP10(2016)142 In the oﬀ-shell formalism the auxiliary ﬁeld T − µν is an antisymmetric tensor, a fun- damental ﬁeld. From this point of view the supersymmetric partners of WµνρσW µνρσ all contain at most two derivatives. This presents a conceptual advantage because it simpliﬁes the initial value problem. On the other hand, in the context of explicit solutions T − µν will coincide with a gauge ﬁeld strength, with one derivative acting on a gauge ﬁeld. We will additionally take D2 = AµνAµν = 0 consistently. Therefore the supersymmetric partners of WµνρσW µνρσ will all represent four-derivative terms on-shell. The coeﬃcient c2 was introduced as the imaginary part of the coupling constant c = c1 + ic2 in the prepotential (3.1). All dependence on the real part c1 has dropped out, because c1 couples only to total-derivative terms such as the Chern-Pontryagin terms ∗WµνρσW µνρσ and Aµν ˜Aµν. We omitted such terms from the Lagrangian since they do not contribute to the equations of motion. 3.2 Equations of motion Many previous studies focused on BPS solutions that preserve the full N = 2 supersym- metry, or at least 1 2-BPS solutions that preserve a residual N = 1 supersymmetry. Such solutions are greatly constrained by relatively simple BPS equations and so it is suﬃcient to consider a small subset of the equations of motion. We are interested in solutions that explicitly break supersymmetry, and so we need to derive and solve the full equations of motion for the Lagrangian (3.2). The only D-dependence in the Lagrangian is the D2 term, and so the D-equation of motion forces D = 0. When combined with the constraint equation (2.16), this forces us to consider solutions with vanishing Ricci scalar R = 0 . (3.3) (3.3) We compute the equations of motion for the matter ﬁelds X, T − µν, Wµ, and Aµ to be, respectively, We compute the equations of motion for the matter ﬁelds X, T − µν, Wµ, and Aµ to be, respectively, 0 = □¯X + 1 2 ¯XR + 1 8 F + µν −1 4XT + µν T +µν , 0 = ¯X F − µν −1 4 ¯XT − µν −c2 2 128D[µDρT + ν]ρ + T − µνT + ρσT +ρσ −64Rρ [µT + ν]ρ , 0 = Dµ F + µν + F − µν −1 2XT + µν −1 2 ¯XT − µν , (3.4) (3.4) 0 = XDµ ¯X −¯XDµX + 8c2 T −µνDρT + ρν −T +µνDρT − ρν −16iDνAµν . 0 = XDµ ¯X −¯XDµX + 8c2 T −µνDρT + ρν −T +µνDρT − ρν −16iDνAµν . – 8 – The ﬁeld strength Fµν must also satisfy the Bianchi identity Dµ ˜Fµν = 0 which we express as he ﬁeld strength Fµν must also satisfy the Bianchi identity Dµ ˜Fµν = 0 which we express as Dµ F + µν −F − µν = 0 . 3.2 Equations of motion In summary, we have shown that any solution to our minimal supergravity theory must satisfy the matter ﬁeld equations of motion (3.4), the Bianchi identity (3.5), the Einstein equation (3.9), and must have a geometry with vanishing Ricci scalar R = 0. 4 Non-supersymmetric solutions In this section we embed arbitrary solutions to Einstein-Maxwell theory into the minimal N = 2 supergravity theory (with a supersymmetrized (Weyl)2 correction) presented in section 3. The matter ﬁelds of the higher-derivative gravity are speciﬁed in terms of the matter in the Einstein-Maxwell theory. The geometry that supports the Einstein-Maxwell solution is unchanged when considered as solution to higher-derivative gravity. JHEP10(2016)142 4.1 Einstein-Maxwell The starting point is the standard Einstein-Maxwell theory LEM = −1 2κ2 R + 1 4FµνFµν , (4.1) (4.1) where κ2 = 8πGN. We are using boldfaced symbols gµν, R , and Fµν for the metric, Ricci scalar, and electromagnetic ﬁeld strength in Einstein-Maxwell theory in order to avoid any confusion with related quantities in the higher-derivative supergravity Lagrangian (3.2). Any solution to Einstein-Maxwell theory satisﬁes the Maxwell equations and the Bianchi identities, which we package together as the Maxwell-Bianchi equations ∇µF± µν = 0 , (4.2) (4.2) where the self-dual and anti-self-dual parts of the ﬁeld strength are deﬁned using the conventions in section 2.2. The geometry and the matter ﬁelds are related by the Ein- stein equation Rµν −1 2gµνR = −F− µρF+ρ ν . (4.3) (4.3) We are particularly interested in Kerr-Newman black hole solutions but our embedding will apply to any solution of Einstein-Maxwell theory. 3.2 Equations of motion (3.5) (3.5) In order to derive the Einstein equation, we ﬁrst rewrite the minimal supergravity La- grangiann (3.2) as L = −1 8π\|X\|2R + L(2) + L(4) , (3.6) (3.6) where L(2) is the Lagrangian for the two-derivative matter terms L(2) = 1 8π 2DµXDµ ¯X + 1 4FµνF µν −1 4Fµν XT +µν + ¯XT −µν + 1 32 X2T + µνT +µν + ¯X2T − µνT −µν , (3.7) (3.7) JHEP10(2016)142 JHEP10(2016)142 while L(4) contains all of the four-derivative terms present in the supersymmetrized Weyl invariant L(4) = 4c2 π WµνρσW µνρσ + 6D2 −2AµνAµν + 1 2(DµT −µν)(DρT + ρν) +1 4Rµ νT − µρT +νρ + 1 512T − µνT −µνT + ρσT +ρσ . (3.8) (3.8) The Einstein equation can now be expressed as The Einstein equation can now be expressed as 1 4π\|X\|2 Rµν −1 2gµνR = T (2) µν + T (4) µν , (3.9) (3.9) where T (2) µν is the energy-momentum tensor for the two-derivative matter T (2) µν = 2 √−g δ √−gL(2) δgµν = 1 4π 2(DµX)(Dν ¯X) −gµν(DρX)(Dρ ¯X) + F + µρF −ρ ν −1 4 XF − µρT +ρ ν + ¯XF + µρT −ρ ν , (3.10) (3.10) while T (4) µν is the energy-momentum tensor for the four-derivative parts of the action while T (4) µν is the energy-momentum tensor for the four-derivative parts of the action T (4) µν = 2 √−g δ √−gL(4) δgµν = 8c2 π 4RµρR ρ ν −gµνRρσRρσ −4 3RµνR + 1 3gµνR2 −2□Rµν + 4DρDµRνρ + 1 3gµν□R −4 3DµDνR −4AµρA ρ ν + gµνAρσAρσ −1 4gµν(DρT − ρτ)(DσT +στ) + 1 2(DµT − νρ)(DσT +σρ) + 1 2(DµT + νρ)(DσT −σρ) + 1 2(DρT − ρµ)(DσT + σν) + 1 1024gµνT − ρσT −ρσT + τλT +τλ −1 8gµνRρσT − ρτT +τ σ + 1 2RµρT − νσT +ρσ + 1 4RρσT − µρT + νσ + 1 4DρDµ(T − νσT +ρσ) −1 8□(T − µρT +νρ) −1 8gµνDρDσ(T −ρτT +σ τ) . (3.1 (3.11) – 9 – In summary, we have shown that any solution to our minimal supergravity theory must satisfy the matter ﬁeld equations of motion (3.4), the Bianchi identity (3.5), the Einstein equation (3.9), and must have a geometry with vanishing Ricci scalar R = 0. 4.2 Embedding Starting from a solution to Einstein-Maxwell theory we specify the matter ﬁelds in the higher-derivative theory as X = √ 4π κ , Aµ = 0 , T ± µν = 4F± µν , F ± µν = 1 4XT ± µν = XF± µν . (4.4) (4.4) As mentioned previously, the geometry is unchanged. As mentioned previously, the geometry is unchanged. The numerical value of X is such that the Ricci scalar term in the Lagrangian (3.2) is normalized correctly 1 L = −1 2κ2 R + . . . . (4.5) (4.5) – 10 – – 10 – By choosing Aµ = 0, the supercovariant derivative operator Dµ reduces to the ordinary i t d i ti t ∇µ By choosing Aµ = 0, the supercovariant derivative operator Dµ reduces to the ordinary covariant derivative operator ∇µ. It is rather straightforward to show that all the matter ﬁeld equations of motion (3.4) are satisﬁed by the matter (4.4). Since F± µν is divergence-free by the Maxwell-Bianchi equations (4.2), T ± µν must be divergence-free as well DµT ± µν = 0 . (4.6) (4.6) Since X is constant and Aµν = 0 the ﬁnal equation in (3.4) follows. We also have DµF ± µν = 0 (since X is constant) and so the gauge ﬁeld equations in the third line of (3.4) are satisﬁed. The scalar equation of motion is satisﬁed because X is constant, the geometry has R = 0, and the matter satisﬁes 1 JHEP10(2016)142 F± µν = F ± µν −1 4XT ± µν = 0 . (4.7) (4.7) The equation of motion for the antisymmetric tensor T − µν is slightly less obvious. It is satisﬁed due to the following identities for (anti-)self-dual tensors in 4D: The equation of motion for the antisymmetric tensor T − µν is slightly less obvious. It is satisﬁed due to the following identities for (anti-)self-dual tensors in 4D: T + µνT −ρσ + T +ρσT − µν = 4δ[ρ [µT + ν]τT −σ]τ , T + µνT −µν = 0 . (4.8) (4.8) At this point we still need to verify the Einstein equation (3.9). It is important to note that the only dependence on c2 is in the four-derivative energy-momentum tensor T (4) µν and not in any of the two-derivative terms. 4.2 Embedding Since we claim the embedding works for any value of the constant c2, the two-derivative and four-derivative terms must cancel independently. The original Einstein equation (3.9) therefore becomes two separate equations 1 4π\|X\|2 Rµν −1 2gµνR = T (2) µν and T (4) µν = 0 . (4.9) (4.9) The energy-momentum tensor T (2) µν , given in (3.10), simpliﬁes greatly due to the embed- ding (4.4). The two-derivative part of the Einstein equations (4.9) becomes The energy-momentum tensor T (2) µν , given in (3.10), simpliﬁes greatly due to the embed- ding (4.4). The two-derivative part of the Einstein equations (4.9) becomes Rµν −1 2gµνR = −F+ µρF−ρ ν . (4.10) (4.10) We recognize this equation as the original condition on the Einstein-Maxwell geome- try (4.3). Taking the trace of this expression yields We recognize this equation as the original condition on the Einstein-Maxwell geome- try (4.3). Taking the trace of this expression yields R = 0 , (4.11) (4.11) R = 0 , as required by the constraint equation (3.3) coming from the auxiliary D-ﬁeld. The four-derivative part of the Einstein equations (4.9), with T (4) µν given in (3.11), becomes 0 = 4RµρR ρ ν −gµνRρσRρσ −4 3RµνR + 1 3gµνR2 −2∇2Rµν + 4∇ρ∇µRνρ + 1 3gµν∇2R −4 3∇µ∇νR + 1 4gµνF− ρσF−ρσF+ τλF+τλ −2gµνRρσF− ρτF+τ σ + 8RµρF− νσF+ρσ + 4RρσF− µρF+ νσ + 4∇ρ∇µ(F− νσF+ρσ) −2∇2(F− µρF+νρ) −2gµν∇ρ∇σ(F−ρτF+σ τ) , (4.12) (4.12) – 11 – upon insertion of the embedding (4.4). It is not immediately obvious that it is realistic to solve this equation. However, repeated use of Rµν = −F+ µρF−ρ ν in (4.12) and careful simpliﬁcation shows that it is in fact satisﬁed identically. In summary, we have veriﬁed that our embedding (4.4) generates a solution to the higher-derivative theory for each solution to the original Einstein-Maxwell theory. This result relies on supersymmetry of the theory, as the action we consider is far from arbitrary. However, the solutions do not generally preserve any supersymmetry. As a check on these results, we consider the special case of extremal Reissner-Nordstr¨om black holes. We have veriﬁed that the BPS equations derived in [11, 12] are satisﬁed by our embedding (4.4) for extremal Reissner-Nordstr¨om geometries. 4.2 Embedding This is expected, since these geometries are known to be 1 2-BPS domain walls that interpolate between the N = 2 supersymmetric AdS2 ×S2 geometry at the horizon and the N = 2 supersymmetric Minkowski spacetime at inﬁnity. JHEP10(2016)142 4.3 Simpliﬁed Lagrangian (4.15) (4.15) – 12 – – 12 – After these simpliﬁcations we ﬁnd After these simpliﬁcations we ﬁnd After these simpliﬁcations we ﬁnd Ltrunc = −1 2κ2 R + 1 4FµνF µν + 4c2 π WµνρσW µνρσ + 4Rµ νF − µρF +νρ + 1 2F − µνF −µνF + ρσF +ρσ + 8(∇µF −µν)(∇ρF + ρν) . (4.16) (4.16) This form of the Lagrangian expresses the intuitive notion that our theory is ordinary Einstein-Maxwell theory with addition of a supersymmetrized Weyl invariant that includes mixings between the electromagnetic ﬁeld strength and the Riemann tensor. Any solution to the truncated theory (4.16) will automatically be a solution to the minimal supergravity theory (3.2). JHEP10(2016)142 Our black hole solutions imply that the supersymmetrized Weyl invariant LN=2 Weyl = WµνρσW µνρσ + 4Rµ νF − µρF +νρ + 1 2F − µνF −µνF + ρσF +ρσ + 8(∇µF −µν)(∇ρF + ρν) (4.17) (4.17) can be included into the Einstein-Maxwell action without consequence to the geometry or the ﬁeld strength. To understand this claim we rewrite WµνρσW µνρσ in terms of the Gauss-Bonnet density E4 as WµνρσW µνρσ = E4 + 2RµνRµν −2 3R2 , (4.18) (4.18) and ﬁnd LN=2 Weyl = E4 + 2 Rµν + F − µρF +ρ ν 2 −2 3R2 + 8(∇µF −µν)(∇ρF + ρν) . (4.19) (4.19) The Gauss-Bonnet density E4 does not contribute to the equations of motion because it is topological. The remaining terms (Rµν + F − µρF +ρ ν )2, R2, and (∇µF −µν)(∇ρF + ρν) are all quadratic in expressions that vanish for Einstein-Maxwell backgrounds. That explains why these terms can be introduced in the Einstein-Maxwell action without changing the original solutions. The simpliﬁcations we ﬁnd are predicated on the precise combination of four-derivative terms appearing in (4.17); any others would lead to complicated corrections of the solutions (see e.g. [16, 17]). In our context those coeﬃcients were dictated by the N = 2 supersym- metry of the theory. Thus supersymmetry is responsible for substantial simpliﬁcations even for solutions that do not preserve any supersymmetry. It was previously noticed in [18] that the entropy of supersymmetric black holes in heterotic string theory is the same whether one introduces higher-derivative corrections in the form of a supersymmetrized Weyl invariant or an ordinary Gauss-Bonnet term. This led to arguments (see e.g. 4.3 Simpliﬁed Lagrangian Having showed that the embedding (4.4) satisﬁes the fairly complicated equations of motion for minimal supergravity with higher-derivative corrections, it is worth understanding why this is the case. We do so by introducing a simpliﬁed eﬀective Lagrangian that captures the same dynamics as the original Lagrangian (3.2) within the context of our embedding. As a ﬁrst step we can consistently eliminate the auxiliary ﬁelds D and Aµ by setting both to zero at the level of the action. We can then use properties of (anti-)self-dual tensors in 4D (4.8) to express the simpliﬁed Lagrangian as 8πLtrunc = −\|X\|2R −1 4FµνF µν + 2∇µX∇µ ¯X + 1 2 F + µν −1 4XT + µν 2 + h.c. + 32c2 WµνρσW µνρσ + 1 4Rµ νT − µρT +νρ + 1 512T − µνT −µνT + ρσT +ρσ + 1 2(∇µT −µν)(∇ρT + ρν) . (4.13) (4.13) We now want to eliminate the auxiliary ﬁelds X and T − µν from the action by replacing them with their ansatz in the embedding (4.4): We now want to eliminate the auxiliary ﬁelds X and T − µν from the action by replacing them with their ansatz in the embedding (4.4): X = √ 4π κ , T − µν = 4 X F − µν , (4.14) (4.14) at the level of the action. We can see from (4.13) that X is sourced by the Ricci scalar, which vanishes for Einstein-Maxwell backgrounds, and F + µν −1 4XT + µν, which vanishes in (4.14). Similarly, T − µν is sourced by F − µν −1 4 ¯XT − µν and various other terms that vanish for Einstein- Maxwell backgrounds. The elimination (4.14) is therefore consistent with the X and T − µν equations of motion and can be implemented at the level of the action. To make the normalization simpler we also rescale the vector multiplet ﬁeld strength by To make the normalization simpler we also rescale the vector multiplet ﬁeld strength by Fµν → √ 4π κ Fµν . 4.3 Simpliﬁed Lagrangian [19]) that the supersymmetrized Weyl invariant should coincide with the Gauss-Bonnet density on-shell. Our supersymmetrized Weyl invariant (4.19) makes this argument concrete. This is particularly surprising in the near-horizon region of BPS black holes: the AdS2 × S2 geometry has vanishing Weyl tensor, yet the supersymmetrized Weyl invariant is non-zero and matches the Gauss-Bonnet density exactly. – 13 – 5 Properties of black holes in higher-derivative gravity In this section we analyze properties of Kerr-Newman black holes considered as solutions to minimal supergravity with higher-derivative corrections. We show that the black hole entropy simpliﬁes when the theory has N = 2 supersymmetry. 5.1 Black hole entropy The black hole entropy in the higher-derivative theory is given by the Wald entropy for- mula [1–3]. The entropy is SWald = 2π Z H δL δRµνρσ ϵµνϵρσ √ h d2x , (5.1) (5.1) JHEP10(2016)142 where hij is the induced metric on the black hole horizon H and ϵµν is the (antisymmetric) unit binormal to the horizon, normalized such that ϵµνϵµν = −2. Four-derivative terms in the action give rise to an integrand that includes terms linear in the curvature and terms with two derivatives acting on the matter ﬁelds. Each of these terms in the integrand is somewhat intricate and upon integration they will generally give complicated contributions to the entropy. However, N = 2 supersymmetry dictates relations between the coeﬃcients of these contributions such that the four-derivative terms combine into the expression (4.19). Any part of the action that is quadratic in terms that vanish on-shell cannot contribute to the Wald entropy (5.1), since the entropy is determined by a linear variation. For the purposes of computing the Wald entropy it is therefore suﬃcient to add the Gauss-Bonnet term LGB = 4c2 π E4 (5.2) (5.2) the standard Einstein-Hilbert Lagrangian. This is a considerable simpliﬁcation. The Gauss-Bonnet term (5.2) is topological, and so any variation of it with respect to physical ﬁelds with produce a total derivative. It can also be expressed in 4D as a total derivative acting on (non-covariant) Christoﬀel symbols. However, the Wald entropy formalism requires ﬁrst putting the Lagrangian in a covariant form, e.g. in terms of the metric and the Riemann tensor. The Wald entropy formalism then requires varying the Lagrangian with respect to the Riemann tensor, not a physical ﬁeld. The contribution to the Wald entropy from a Gauss-Bonnet term in the action is therefore not forced to be zero. This contribution has been studied in detail; it was explicitly shown by Wald and Iyer in [2] that this contribution is proportional to the Euler characteristic of the surface of integration. This result was also independently shown in [20] by use of the Jacobson-Myers functional. The total Wald entropy, including the area law due to the Einstein-Hilbert action, is SWald = AH 4GN + 128πχ(2)c2 , (5.3) (5.3) where χ(2) is the Euler characteristic of the black hole horizon1 χ(2) = −1 4π Z H dA R(2) . 5.1 Black hole entropy (5.4) (5.4) 1Our curvature conventions are set by the sign on the Ricci scalar in the Einstein-Hilbert action. The curvature of a sphere is negative and the Euler character (5.4) has an unusual overall minus sign. 1Our curvature conventions are set by the sign on the Ricci scalar in the Einstein-Hilbert action. The curvature of a sphere is negative and the Euler character (5.4) has an unusual overall minus sign. – 14 – For general Kerr-Newman black holes, the Euler characteristic of the horizon is χ(2) = 2, and so the Wald entropy (5.3) becomes For general Kerr-Newman black holes, the Euler characteristic of the horizon is χ(2) = 2, and so the Wald entropy (5.3) becomes SWald = AH 4GN + 256πc2 . (5.5) (5.5) This is the entropy of a Kerr-Newman black hole, including the higher-derivative correction in the form of a supersymmetrized Weyl invariant. In the special case of vanishing charge, the black hole geometry is Ricci ﬂat Rµν = 0 and so it is obvious that the Weyl invariant coincides with the Gauss-Bonnet term on-shell. We ﬁnd that this well-known statement generalizes to Kerr-Newman black holes. That is interesting because this family includes a BPS limit, where the black hole preserves the supersymmetry of the theory and the microscopic description is under control. Previous studies [4–6, 9–11, 18, 21–26] have found that higher-derivative corrections in string theory gives rise to a correction of the form (5.5) with a numerical coeﬃcient that can be matched with microscopic considerations. JHEP10(2016)142 Our result for the correction to the black hole entropy (5.5) has no dependence what- soever on the parameters of the black hole. The deformation away from the BPS limit by adding mass and introducing angular momentum does not change the correction due to higher-order derivatives. This is reminiscent of our previous result [7] that quantum corrections to Kerr-Newman black holes are universal and similarly insensitive to defor- mations oﬀextremality. For both classes of corrections it is signiﬁcant that the theory preserves N = 2 supersymmetry but it is unimportant whether the black holes preserve the supersymmetry of the theory. 5.2 OSV conjecture The correction to the entropy due to the higher-derivative terms is just a constant, in- dependent of the black hole parameters. The value of the constant is therefore captured by the BPS limit and so it can be interpreted in string theory, e.g. following the OSV conjecture [27]. For extremal BPS black holes, the attractor mechanism [28–31] speciﬁes scalars in the horizon AdS2 × S2 geometry in terms of the charges (pI, qI) by the attractor equations pI = Re[CXI] , (5.6) qI = Re[CFI] , (5.7) (5.6) (5.7) where C is an arbitrary scaling parameter chosen as where C is an arbitrary scaling parameter chosen as C2 ˆA = 256 , (5.8) (5.8) th ˆA evaluated at the horizon. Expressing the real and imaginary parts of the scalars as with ˆA evaluated at the horizon. Expressing the real and imaginary parts of the scalars as i CXI = pI + i πφI , (5.9) (5.9) the black hole potential is the black hole potential is F(φI, pI) = −π Im[C2F(XI, ˆA)] , (5.10) (5.10) – 15 – in a mixed ensemble deﬁned as a microcanonical ensemble of magnetic charges pI and a canonical ensemble of electric charges qI with chemical potentials φI. The black hole entropy, including higher-derivative terms, is then given by the Legendre transform in a mixed ensemble deﬁned as a microcanonical ensemble of magnetic charges pI and a canonical ensemble of electric charges qI with chemical potentials φI. The black hole entropy, including higher-derivative terms, is then given by the Legendre transform SBH(qI, pI) = 1 −φI ∂ ∂φI F(φI, pI) , (5.11) (5.11) where the electric potentials φI have been eliminated in favor of the electric charges qI through the attractor equation (5.7). In the case of our minimal prepotential (3.1) the attractor equations are In the case of our minimal prepotential (3.1) the attractor equations are JHEP10(2016)142 p = Re[CX] , q = Im[CX] = 1 πφ , (5.12) (5.12) and the black hole potential (5.10) becomes F(φ, p) = π 2 p2 −1 2πφ2 + 256πc2 . (5.13) (5.13) The Legendre transform of this potential gives the black hole entropy SBH = π 2 (q2 + p2) + 256πc2 . 6 Discussion The motivation for studying Kerr-Newman black holes in string theory is the hope that a precision understanding can be achieved in this setting. We are still far from that goal but we can make some observations in the spirit of phenomenology. The classical black hole entropy of Kerr-Newman black holes computed from the outer and inner horizons is S± = 2π M2 −1 2Q2 ± p M2(M2 −Q2) −J2 . (6.1) (6.1) JHEP10(2016)142 An appealing (but speculative) interpretation of these formulae identifes the combinations SR = 1 2(S+ + S−) , SL = 1 2(S+ −S−) , (6.2) (6.2) with the entropy of factorized right- and left-moving excitations of an underlying CFT with (0, 4) supersymmetry [36–38]. This theory would be a generalization of the MSW CFT describing the BPS and near-BPS limits [39]. The assignment of supersymmetry is such that the dependence on the angular momentum quantum number can be entirely accounted for by an SU(2)R current, arbitrarily far from extremality. This is analogous to the standard BMPV model of rotating BPS black holes in ﬁve dimensions [40, 41]. The correction to the black hole entropy due to higher-derivative terms (5.5) is not just independent of black hole parameters; it is the same when computed at the outer and the inner horizons [42]. Therefore, the prescription (6.2) with higher-derivative corrections included identiﬁes the corrections as pertaining to the “Right” sector, with no corrections in the “Left” sector. The “Left” sector contains the novel excitations, the ones that BPS conditions force into their ground state. These are also the ones that carry the angular momentum of the black hole so the BPS limit is incompatible with rotation. The independence of corrections on black hole parameters suggest that this sector receives no string corrections in the leading approximation. At the level of a phenomenological model this is not unreasonable since, after all, the “Left” sector is subject to N = 4 supersymmetry, albeit spontaneously broken by the state. It would clearly be interesting to develop such a model in more detail. Acknowledgments We thank Pedro Lisb˜ao and Daniel R. Mayerson for useful discussions and encouragement. This work was supported in part by the U.S. Department of Energy under grant DE-FG02- 95ER40899. 5.2 OSV conjecture (5.14) (5.14) The ﬁrst term agrees with the classical area law for an extremal Reissner-Nordstr¨om black hole with dyonic U(1) charge, and the correction agrees with our result (5.5) computed using the Wald entropy formalism. The ﬁrst term agrees with the classical area law for an extremal Reissner-Nordstr¨om black hole with dyonic U(1) charge, and the correction agrees with our result (5.5) computed using the Wald entropy formalism. The OSV conjecture [27] makes a connection with microscopic considerations through the relation ZBH = \|Ztop\|2 , (5.15) ZBH = \|Ztop\|2 , (5.1 (5.15) where ZBH is the supersymmetric partition function where ZBH is the supersymmetric partition function where ZBH is the supersymmetric partition function ZBH(φ, p) = exp F(φ, p) (5.16) (5.16) of a four-dimensional BPS black hole in the mixed ensemble. The partition function of the topological string is similarly of a four-dimensional BPS black hole in the mixed ensemble. The partition function of the topological string is similarly Ztop(φ, p) = exp Ftop(φ, p) , (5.17) (5.17) with with with Ftop(λ, X) = X g=0 λ2g−2 top Ftop,g(X) , (5.18) Ftop(λ, X) = X g=0 λ2g−2 top Ftop,g(X) , (5.18) (5.18) a perturbative expansion in the coupling constant λtop = 4πi p+iq. The correction we consider is charge-independent, corresponding to the torus partition function with genus g = 1. a perturbative expansion in the coupling constant λtop = p+iq. The correction we consider is charge-independent, corresponding to the torus partition function with genus g = 1. The OSV conjecture and its possible extensions have been subject to much study and debate, including [26, 32–35]. Since the minimal model we consider has nV = 0 moduli it corresponds to a somewhat singular limit, the case of a rigid Calabi-Yau (in the language of the A-model). It would be interesting to study this special case further. – 16 – A Oﬀ-shell N = 2 supergravity In this section, we summarize the construction of N = 2 supergravity in 4D following the oﬀ-shell formalism studied in [10, 11, 13–15]. We review the bosonic ﬁeld content and – 17 – Weyl Multiplet Vector Multiplet Field gµν bµ Aµ V i µ j T − µν D XI W I µ Y I ij Weyl weight −2 0 0 0 −1 2 1 0 2 Chiral weight 0 0 0 0 −1 0 −1 0 0 Table 3. Bosonic content of the Weyl and vector multiplets, with the corresponding Weyl and chiral weights. Weyl Multiplet Vector Multiplet Field gµν bµ Aµ V i µ j T − µν D XI W I µ Y I ij Weyl weight −2 0 0 0 −1 2 1 0 2 Chiral weight 0 0 0 0 −1 0 −1 0 0 Table 3. Bosonic content of the Weyl and vector multiplets, with the corresponding Weyl and chiral weights. able 3. Bosonic content of the Weyl and vector multiplets, with the corresponding Weyl and iral weights. JHEP10(2016)142 discuss actions that realize the full N = 2 supersymmetry with higher-derivative corrections present. These steps justify the Lagrangian (2.15) that we use to study non-supersymmetric solutions of higher-derivative supergravity. A.1 N = 2 supergravity multiplets The ﬁrst step in constructing oﬀ-shell N = 2 supergravity is to build up an N = 2 super- conformal gauge theory. We then turn this into a theory of supergravity by realizing the superconformal symmetries as spacetime symmetries (instead of internal symmetries). The Weyl multiplet is the multiplet that contains all of the gauge ﬁelds of these superconformal transformations, as well as some auxiliary ﬁelds that must be added for consistency. The bosonic content of the Weyl multiplet includes the metric gµν, the dilatation generator bµ, the SU(2)R gauge ﬁeld V i µ j (where i and j are SU(2) indices), the U(1)R gauge ﬁeld Aµ, the auxiliary anti-self-dual antisymmetric tensor T − µν and the auxiliary real scalar D. µ We will couple this Weyl multiplet to nV + 1 oﬀ-shell vector multiplets, indexed by I = 0, . . . , nV . The bosonic content of each vector multiplet is a complex scalar XI, a U(1) gauge ﬁeld W I µ, and an auxiliary SU(2) triplet of real scalars Y I ij. We summarize the bosonic ﬁeld content of the Weyl and vector multiplets in table 3, as well as the Weyl and chiral weights of each of the ﬁelds. We will also couple our theory to a chiral multiplet. We will eventually identify the ﬁelds in the chiral multiplet with various contractions of ﬁelds from the Weyl multiplet, in order to introduce higher-derivative corrections to the theory. For now, we will keep the chiral multiplet fully general. The bosonic content of this multiplet includes the complex scalars ˆA and ˆC, a complex SU(2) triplet of scalars ˆBij, and an anti-self-dual tensor ˆF − µν. The Weyl and chiral weights w and c of the lowest-component scalar ˆA are arbitrary, but we can express the weights of the other ﬁelds in terms of these weights, as shown in table 4. A.2 Oﬀ-shell action The interactions between the Weyl multiplet and the matter ﬁelds in the vector and chiral multiplets are conveniently summarized by introducing a prepotential F ≡F(XI, ˆA), a meromorphic function of the vector multiplet scalars XI and the chiral multiplet scalar ˆA. Derivatives of the prepotential are denoted by ∂F ∂XI = FI , ∂F ∂ˆA = FA . (A.1) (A.1) – 18 – Chiral Multiplet Field ˆA ˆBij ˆF − µν ˆC Weyl weight w w + 1 w + 1 w + 2 Chiral weight c c + 1 c + 1 c + 2 Table 4. Bosonic content of the chiral multiplet, with arbitrary Weyl and chiral weights w and c for the lowest-component scalar ˆA. Table 4. Bosonic content of the chiral multiplet, with arbitrary Weyl and chiral weights w and c for the lowest-component scalar ˆA. Table 4. Bosonic content of the chiral multiplet, with arbitrary Weyl and chiral weights w and c for the lowest-component scalar ˆA. JHEP10(2016)142 The prepotential is holomorphic and does not depend on the complex conjugate scalars ¯XI and ¯ˆA, and so F¯I = F ¯ A = 0. The prepotential is also homogeneous of second degree with respect to Weyl-weighted scalings of XI and ˆA, so F(λXI, λw ˆA) = λ2F(XI, ˆA) , (A.2) (A.2) where w is the Weyl weight of the chiral multiplet scalar ˆA and λ is some arbitrary scaling constant. An equivalent statement of this homogeneity is FIXI + wFA ˆA = 2F . (A.3) (A.3) The action is S = Z d4x √−g L , (A.4) (A.4) where L is the Lagrangian for our oﬀ-shell theory. The purely bosonic part of the La- grangian that couples the Weyl multiplet, the vector multiplets, and the chiral multiplet via interactions dictated by the prepotential is 8πL = iDµFIDµ ¯XI −iFI ¯XI 1 6R −D −i 8FIJY I ijY Jij + i 4FIJ F −I µν −1 4 ¯XIT − µν F −µνJ −1 4 ¯XJT −µν −i 8FI F +I µν −1 4XIT + µν T +µν −i 32FT + µνT +µν + i 2FAI F −I µν −1 4 ¯XIT − µν ˆF −µν + i 2FA ˆC −i 8FAA εikεjl ˆBij ˆBkl −2 ˆF − µν ˆF −µν −i 4FAI ˆBijY Iij + h.c. A.2 Oﬀ-shell action , (A.5) (A.5) where the (bosonic) supercovariant derivative acts on the vector multiplet scalars XI and the chiral multiplet scalar ˆA by where the (bosonic) supercovariant derivative acts on the vector multiplet scalars XI and the chiral multiplet scalar ˆA by DµXI = (∂µ −bµ + iAµ)XI , Dµ ˆA = (∂µ −wbµ −icAµ) ˆA . (A.6) (A.6) The Lagrangian (A.5) has a term linear in the auxiliary D ﬁeld 8πL = i(FI ¯XI −¯FIXI) D −1 6R + . . . , (A.7) (A.7) – 19 – which leads to inconsistent equations of motion. In order to ﬁx this, we can couple a non-linear multiplet to the Lagrangian such that all linear terms in D are cancelled. The bosonic content of this non-linear multiplet includes two SU(2) scalar ﬁelds Φi α, where i is the SU(2) index and α = 1, 2, a real vector ﬁeld Vµ, and a complex antisymmetric matrix Mij of scalars. Ignoring all fermionic terms, the non-linear multiplet is subject to the constraint DµVµ −1 2V µVµ −1 4\|Mij\|2 + DµΦi αDµΦα i = D + 1 3R , (A.8) (A.8) where DµVµ indicates the bosonic supercovariant derivative acting on Vµ. In order to cancel the linear D-dependence in (A.5), we must add the term JHEP10(2016)142 i(FI ¯XI −¯FIXI) DµVµ −1 2V µVµ −1 4\|Mij\|2 + DµΦi αDµΦα i −D −1 3R (A.9) i(FI ¯XI −¯FIXI) DµVµ −1 2V µVµ −1 4\|Mij\|2 + DµΦi αDµΦα i −D −1 3R (A.9) (A.9) to the Lagrangian. The resulting bosonic Lagrangian is 8πL = −i 2(FI ¯XI −¯FIXI)R + iDµFIDµ ¯XI −i 8FIJY I ijY Jij + i 4FIJF−I µν F−µνJ −i 8FIF+I µν T +µν −i 32FT + µνT +µν + i 2FAIF−I µν ˆF −µν + i 2FA ˆC −i 8FAA εikεjl ˆBij ˆBkl −2 ˆF − µν ˆF −µν −i 4FAI ˆBijY Iij + h.c. + i(FI ¯XI −¯FIXI) DµVµ −1 2V µVµ −1 4\|Mij\|2 + DµΦi αDµΦα i , (A.10) (A.10) where we have deﬁned the supercovariant ﬁeld strengths where we have deﬁned the supercovariant ﬁeld strengths where we have deﬁned the supercovariant ﬁeld strengths F+I µν = F +I µν −1 4XIT + µν , F−I µν = F −I µν −1 4 ¯XIT − µν . (A.11) (A.11) B Simplifying the Lagrangian JHEP10(2016)142 In this section, we will simplify the Lagrangian (A.10) both by partially gauge-ﬁxing our theory and by eliminating various auxiliary ﬁelds via their equations of motion. A.3 Higher-derivative interactions At this point the bosonic Lagrangian (A.10) contains the Ricci scalar but no higher- derivative gravity terms. One way to introduce these is to identify the chiral multiplet ﬁelds with various contractions of ﬁelds in the Weyl tensor, chosen precisely such that the supersymmetry variations are all consistent. Roughly speaking, we set the chiral multiplet to be the square of the Weyl multiplet. Ignoring all fermionic terms, this identiﬁes the chiral multiplet ﬁelds as ˆA = T − µνT −µν , ˆBij = −16εk(iV k µν j)T −µν , ˆF − µν = −16 WµνρσT −ρσ + DT − µν + 2iAρ[µT −ρ ν] , ˆC = 32 WµνρσW µνρσ + i∗WµνρσW µνρσ + 6D2 −2AµνAµν + 2Aµν ˜Aµν −1 2T −µνDµDρT + ρν + 1 4Rµ νT − µρT +νρ + 1 256T − µνT −µνT + ρσT +ρσ +1 2V i µν jVµνj i −1 2V i µν j ˜Vµνj i , (A.12) µ ˆBij = −16εk(iV k µν j)T −µν , ˆF − µν = −16 WµνρσT −ρσ + DT − µν + 2iAρ[µT −ρ ν] , (A.12) – 20 – where the ﬁeld strength of the SU(2)R gauge ﬁeld V i µ j is where the ﬁeld strength of the SU(2)R gauge ﬁeld V i µ j is V i µν j = ∂µV i ν j −∂νV i µ j + 1 2V i µ kV k ν j −1 2V i ν kV k µ j . (A.13) (A.13) The scalar ˆA now has Weyl weight w = 2 and chiral weight c = −2. The bosonic Lagrangian (A.10) still retains the same form, but this identiﬁcation introduces higher- derivative interactions to the theory that we are interested in studying. B.1 Partial gauge-ﬁxing The Lagrangian (A.10) has an N = 2 superconformal symmetry that acts as a gauge sym- metry. To obtain an N = 2 Poincar´e supergravity theory, we must gauge-ﬁx the extra gauge symmetries of the superconformal theory, including special conformal transforma- tions, dilatations, and a local chiral SU(2)R × U(1)R symmetry. We gauge-ﬁx the special conformal symmetry by choosing the K-gauge bµ = 0 . (B.1) (B.1) To gauge-ﬁx the dilatational symmetry, we choose the D-gauge that sets the K¨ahler po- tential to be constant 8 To gauge-ﬁx the dilatational symmetry, we choose the D-gauge that sets the K¨ahler po- tential to be constant 8 e−K ≡i(FI ¯XI −¯FIXI) = 8π κ2 , (B.2) (B.2) with the value of the constant chosen to reproduce the standard normalization of the Einstein-Hilbert term in the action. The local chiral SU(2)R invariance can be gauge-ﬁxed by imposing the V -gauge with the value of the constant chosen to reproduce the standard normalization of the Einstein-Hilbert term in the action. The local chiral SU(2)R invariance can be gauge-ﬁxed by imposing the V -gauge Φi α = δi α . (B.3) (B.3) Finally, to gauge-ﬁx the local chiral U(1)R symmetry, we choose the A-gauge X0 = ¯X0 . (B.4) (B.4) Note that the D-gauge (B.2) and A-gauge (B.4) remove two degrees degree of freedom from the vector multiplet scalars, and thus the Poincar´e supergravity theory has only nV independent scalars. Note that the D-gauge (B.2) and A-gauge (B.4) remove two degrees degree of freedom from the vector multiplet scalars, and thus the Poincar´e supergravity theory has only nV independent scalars. at the level of the action. We now want to eliminate the non-linear multiplet ﬁelds Vµ and Mij from (A.10), sub- ject to the constraint (A.8). These non-linear multiplet ﬁelds interact with the other matter ﬁelds only through the K¨ahler potential e−K = i(FI ¯XI −¯FIXI), which is set to a constant via the D-gauge condition (B.2). The non-linear multiplet ﬁelds eﬀectively decouple from the rest of our theory, and so we can study their equations of motion independently from the others. We ﬁnd that we can choose JHEP10(2016)142 Vµ = 0 , Mij = 0 , (B.6) (B.6) at the level of the action, as long as the background value of D satisﬁes D = −1 3R. B.2 Eliminating auxiliary ﬁelds The remaining independent auxiliary ﬁelds in our theory are the SU(2)R gauge ﬁeld V i µ j, the U(1)R gauge ﬁeld Aµ, the vector multiplet SU(2) triplets Y I ij, the non-linear multiplet ﬁelds Vµ and Mij, and the anti-self-dual antisymmetric tensor T − µν. We will use the auxiliary equations of motion to eliminate everything except Aµ and T − µν from the action. – 21 – If we insert the chiral multiplet ﬁeld expressions (A.12) into the Lagrangian (A.10), we ﬁnd that the ﬁelds Y I ij and V i µ j and their derivatives appear at least quadratically with one another in the action. It is therefore consistent to set them both to zero Y I ij = 0 , V i µ j = 0 , (B.5) (B.5) at the level of the action. References [1] R.M. Wald, Black hole entropy is the Noether charge, Phys. Rev. D 48 (1993) R3427 [gr-qc/9307038] [INSPIRE]. [2] V. Iyer and R.M. Wald, Some properties of Noether charge and a proposal for dynamical black hole entropy, Phys. Rev. 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(B.8) (B.8) Since the SU(2)R gauge ﬁeld V i µ j is set to zero, the chiral multiplet ﬁelds from (A.12) become Since the SU(2)R gauge ﬁeld V i µ j is set to zero, the chiral multiplet ﬁelds from (A.12) become ˆA = T − µνT −µν , ˆ ˆA = T − µνT −µν , ˆBij = 0 , ˆF − µν = −16 WµνρσT −ρσ + DT − µν + 2iAρ[µT −ρ ν] , ˆC = 32 WµνρσW µνρσ + i∗WµνρσW µνρσ + 6D2 −2AµνAµν + 2Aµν ˜Aµν −1 2T −µνDµDρT + ρν + 1 4Rµ νT − µρT +νρ + 1 256T − µνT −µνT + ρσT +ρσ . (B.9) ˆA = T − µνT −µν , (B.9) If we take the full Lagrangian (A.10) and implement the consistent truncation (B.7), we ﬁnd If we take the full Lagrangian (A.10) and implement the consistent truncation (B.7), we ﬁnd 8πL = −i 2(FI ¯XI −¯FIXI)R + iDµFIDµ ¯XI + h.c. + i 4FIJF−I µν F−µνJ −i 8FIF+I µν T +µν −i 32FT + µνT +µν + i 2FAIF−I µν ˆF −µν + i 2FA ˆC + i 4FAA ˆF − µν ˆF −µν + h.c. . (B.10) + i 4FIJF−I µν F−µνJ −i 8FIF+I µν T +µν −i 32FT + µνT +µν + i 2FAIF−I µν ˆF −µν + i 2FA ˆC + i 4FAA ˆF − µν ˆF −µν + h.c. . 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https://openalex.org/W3046961276	https://www.e3s-conferences.org/articles/e3sconf/pdf/2020/43/e3sconf_i2cnp2020_04004.pdf	English	null	Chemical characterization of almond meal as a co-product of the mechanical extraction of almond oil	E3S web of conferences	2,020	cc-by	6,458	Chemical characterization of almond meal as a co-product of the mechanical extraction of almond oil. Nadia Houmy1,5, Reda Melhaoui1, Kamal Belhaj1, Aurore Richel2, Marianne Sindic3, Christophe Hano4, Souhayla Kodad1, Aatika Mihamou1,Mohamed Addi1, Malika Abid1 and Ahmed Elamrani1. 1 Laboratory for Agricultural Productions Improvement, Biotechnology and Environment, Faculty of Sciences, University of Mohammed Premier, Oujda, Morocco y j 2 Laboratory of Biological and Industrial Chemistry, University of Liège, Gembloux, Belgium 3 Laboratory QSPA, Gembloux Agro Bio Tech, University of Liège, Gembloux, Belgium 4Laboratoire de Biologie des Ligneux et des Grandes Cultures, OrleansUniversity, France 5 5 Laboratory of Food Technology and Quality, CRRA, INRA, Oujda, Morocco. 5 Laboratory of Food Technology and Quality, CRRA, INRA, Oujda, Morocco. Abstract.Almonds harvesting, along with precleaning, hulling, shelling, and sorting, generate recoverable co-products such as double, malformed, or damaged almonds. The common way that generates more benefit is the extraction of almond oil for food and cosmetic uses. The aim of this study is to compare composition and nutritional value of almond meals of the main varieties from eastern Morocco, Marcona (M), Fournat de Breznaud (FNB), FerragnesandFerraduel (FF) and Beldi a local ecotype (B), during three consecutive crop years (2016-2018). Significant differences were observed, which mainly concern the residual oil in almond cakes and especially their fibres, sugars, and proteins. Differences depend on the quality of the raw material and the effects of the crop year on the variety. Besides, from a nutritional point of view, analysed almond meals still contain significant amounts of residual oil and the major constituents of almond seeds (sugars, fibres, and proteins). The amino acid profile presents eight essential amino acids (Cysteine, Methionine, Isoleucine, Leucine, Phenylalanine, Tyrosine, and Lysine Valine) and the Protein Digestibility Corrected Amino Acid Score (PDCAAS) ranges between 24.78% for (M) and 62.17% for (FF). We conclude the analysed almond meals present interesting nutritional values as a specific ingredient for gluten-free culinary preparations and light foodstuffs. E3S Web of Conferences 183, 04004 (2020) I2CNP 2020 E3S Web of Conferences 183, 04004 (2020) I2CNP 2020 E3S Web of Conferences 183, 04004 (2020) https://doi.org/10.1051/e3sconf/202018304004 Corresponding author:houmy.nadia@gmail.com 1 Introduction very rich in fat, making them a perfect source of edible oil and oil for cosmetic [3]. In eastern Morocco, thanks to the Moroccan green plan and the PROFAO (Solidarity farming Project of Almond Sector in Eastern Morocco, 2011-2018); 6000 hectares of new almond orchards were created. PROFAO, as an integrated project, allows the organisation of almond's farmer cooperatives and facilitates the creation of economic interest grouping for almond post-harvest facilities, processing, production and marketing of almonds and derivatives [4]. Almond nuts crushing, shelling and sorting operations generate co-products like doubles malformed almonds or mechanically damaged (split and broken kernels) and defects seeds. The best way for exploiting this co-product and deriving more benefits and add value is the mechanical extraction of almond oil for food and cosmetic uses. This process of almond oil extraction also generates another by-product (Almond residue or almond meal) which represents at least 50% by gross mass of the raw material of damaged almonds. Thus, the aim of this study is to assess the nutritional value of these almond residues by analysing the chemical composition of almond meals from fourintroduced varieties, Marcona (M), Fournat de Breznaud(FNB), FerragnesandFerraduel(FF) and Beldi a local ecotype (B), during three consecutive crop years (CY1: 2016; CY2: 2017; CY3: 2018). 1 Introduction Almonds are the edible seeds of the Prunus amygdalus Dulcis tree, more commonly known as the almond tree, which is native from its wild ancestors of the central plateau of Asia, currently domesticated, and widely cultivated in Mediterranean climate areas [1]. Although Corresponding author:houmy.nadia@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/). E3S Web of Conferences 183, 04004 (2020) I2CNP 2020 https://doi.org/10.1051/e3sconf/202018304004 almonds are commonly referred to as nuts, they are actually the seeds found at the centre of the almond fruit. Almond fruit consists of four parts: kernel or Seed or "meat", seed coat (a thin leathery layer known as brown skin of the seed), almond shell, and almond hull which corresponds to the outer green shell cover [2]. almonds are commonly referred to as nuts, they are actually the seeds found at the centre of the almond fruit. Almond fruit consists of four parts: kernel or Seed or "meat", seed coat (a thin leathery layer known as brown skin of the seed), almond shell, and almond hull which corresponds to the outer green shell cover [2]. The kernel or seed is the edible part of the nut, with a high nutritional value. Almond kernels can be consumed raw or cooked, blanched, ground into flour and even made into non-dairy milk. It can be also incorporated, as an ingredient, into culinary recipes. They are very rich in fat, making them a perfect source of edible oil and oil for cosmetic [3]. The kernel or seed is the edible part of the nut, with a high nutritional value. Almond kernels can be consumed raw or cooked, blanched, ground into flour and even made into non-dairy milk. It can be also incorporated, as an ingredient, into culinary recipes. They are very rich in fat, making them a perfect source of edible oil and oil for cosmetic [3]. In eastern Morocco, thanks to the Moroccan green plan and the PROFAO (Solidarity farming Project of Almond Sector in Eastern Morocco, 2011-2018); 6000 hectares of new almond orchards were created. PROFAO, as an integrated project, allows the organisation of almond's farmer cooperatives and facilitates the creation of economic interest grouping for almond post-harvest facilities, processing, production and marketing of almonds and derivatives [4]. 2.1 Plant material and sampling In eastern Morocco, producers and processors of almonds in the Sidi Bouhria region are organised into an economic interest grouping (GIE- Sidi Bouhria). In order to derive maximum benefit from their almond production with the perspective of zero waste and using everything the almonds orchard grows. Thus, the almond kernels grading and sorting co-product, corresponding to split & broken malformed and double almonds, is intended for the extraction of almond oil for food and cosmetics. In addition to almond oil, the mechanical process produces a residue “the almond cake /"meal". This study concerns the chemical characterisation of the almond meals of the five most cultivated varieties in the region of eastern Morocco. The almonds come from five-year-old almond trees, installed on a limestone soil located in Sidi Bouhria, in the east of Morocco (GPS coordinates: 34° 44ʹ13.6ʹ'N, 002° 20ʹ15.0ʹ' W). The orchards are rain-fed with a minimum of additional irrigation under semi-arid climatic conditions (Table 1). The GIE-Sidi-Bouhria provided the almond meals used in this study. Table 1. The average temperature and annual cumulated precipitation registered at the closest meteorological station to Sidi Bouhria region (GPS coordinates: 34° 44ʹ13.6ʹ'N, 002° 20ʹ15.0ʹ' W) during the three crop years (CY) 2016-2018 Sidi Bouhria crop year Average temperature (°C) Annual cumulated precipitation(mm) Minimum Maximum CY1 (2016) 11.5 24.5 289.6 CY2 (2017) 12.5 25.6 231.5 CY3 (2018) 12 26.0 205.4 2 2 E3S Web of Conferences 183, 04004 (2020) I2CNP 2020 E3S Web of Conferences 183, 04004 (2020) https://doi.org/10.1051/e3sconf/202018304004 2.3.2 Residual oil Residual oil in almond meal of deferent varieties was performed by chemical extraction using a Soxhlet apparatus, where 200 ml of hexane was used for a total oil extraction of 5g of powdered almond meal then the solvent was evaporated under vacuum using a rotary evaporator and the oil was collected [6]. 2.3.1 Dry matter Dry matterwas carried out in accordance with the thermogravimetric method [5]. It consists to dry 5 to 20 g of the homogenized sample at 103° ± 2° C until obtaining a constant weight. 2.2 Mechanical extraction of almond oil The mechanical extraction of almond oil was carried out by a mechanical press system (IBG MonfortsOekotec GmbH & Co.KG, made in Germany). The screw speed was 60 RPM, and the fine particles were removed from the almond oil by filtration followed by centrifugation at 3000 RPM for 15min. The GIE-Sidi Bouhria provided almond meal samples for the Physico-chemical analyses. Samples were crushed and sieved through a 1 mm mesh sieve, then stored at 4°C until use. Analyses were made in triplicate for the almond meal of each variety. 2.3.3 Total sugars Total sugars contentsis determined according to the Bertrand method with little modifications in four main steps:The first step performed by acid hydrolysis and neutralization of the entire hydrolysate to transform all the polysaccharides into simple reducing sugars. The second step is a sample deproteinisation. The third step consistinto precipitation and filtrationof sugars extract. Finally, titration with potassium permanganate was carried out for determination of total sugar contents in analysed samples of almond meal. The result is expressed as a percentage of mass of glucose in 100 g of sample [7]. 2.3.4 Dietary fibres The determination of dietary fibreswas carried out according to the AOAC 991 method [8]. Three successive enzymatic attacks were carried out (α-amylase, protease and α - amyloglucosidase) to eliminate starch and proteins. The fibresare precipitated by technical ethanol. The obtained value was corrected by determining the residual ash (Muffle furnace), proteins (Kjeldahl) and by the blank. 2.3.5 Ash Ash is determined according to the AACC 2001method [9]. The aim is to determine the mineral content of the sample by heating and so destroying the organic material. Thus, 5 g of the already dried sample was introduced in a pre-tared crucible. Then the sample was introduced into a Muffle furnace at 550 °C overnight, then allowed to cool in a desiccator and finally the weight difference was measured. 3 3 3 E3S Web of Conferences 183, 04004 (2020) I2CNP 2020 https://doi.org/10.1051/e3sconf/202018304004 2.3.7 Amino acid profiles The determination of amino acids by HPLC is carried out according to the method of Spackman et al., [12] by acid hydrolysis of almond meal proteins. This analysis depends on the protein content in sample (500 mg for a sample containing 2% of nitrogen). In our case, 130 mg of each almond meal sample was taken and dissolved in 10 ml of 6 N-HCl containing 0.1% phenol. The samples were mixed and sealed in the presence of nitrogen, and then were heated in oven at 110° C for 24 hours. After cooling in ice, a solution of 30 ml of a citrate buffer at pH 2.2 was added, then a pH adjustment was carried out with 7.5 N and 1 N of NaOH respectively in order to obtain a pH of 2.2. The solutions thus obtained were diluted with citrate buffer (pH = 2.2), after adding 1 ml of Norleucine at 50 μM / ml as internal standard. The solutions were partially filtered by 0.2 µm filter. A 20-µl of aliquot was injected into the HPLC using a sodium oxidase column, cation exchange resin, a column post for the derivation of amino acids into ninhydrin and a UV detector at 570 nm except for Proline at one length 440 nm wave. 2.3.6 Proteins Total nitrogen content was estimated by the Kjeldahl method [10], then protein content was calculated by multiplying the total nitrogen content by a conversion factor of 5.18 [11]. AA: amino acid (g of 100 g of protein) 3 Results and discussion Almond processing coproducts might represent a precious source of raw materials for food, cosmetic, and pharmaceutical industries, however, their valorisation needs a fine physicochemical characterization for the development of novel procedures to be implemented to the sectorial industries. Indeed, providing innovative products from Almond pressed-oil residues (communally named almond oilcake or almond meal) could provide benefit to the overall almond production. Table 3 shows chemical composition of almond meals, corresponding to almond pressed-oil residue, of five most cultivated varieties in the regions of eastern Morocco [Marcona (M), Fournat de Breznaud (FNB), Ferragnes and Ferraduel (FF) and the Beldi (B)]. These results (table 3) concern three consecutive crop years and six analysed parameters of Almond meals: dry matter, residual oil, sugars, fibres,ash andproteins contents.In general, the chemical composition of almond oil meal change between varieties and crop years mainly for dry matter, sugars and ash. 2.3.8 Protein quality calculations The protein quality evaluation was studied by calculation of sum essential amino acid (ΣEAA), sum of non-essential amino acid (ΣNEAA) (indicate the importance of the nutritional value of proteins), Chemical Index (CI) and Proteins Digestibility of Corrected Amino Acid Score (PDCAAS). This later has been adopted as the chosen method for the measurement of the protein value in human nutrition [13]. p The Chemical Index (CI) was calculated on the basis of the procedure described previously by Sujak et al (2006),based on following calculation[(% limiting amino acid)sample/(%limiting amino acid)protein of reference)]x100 [14]. In our case, limiting amino acidswere Met + Cys and the protein referenceused of amino acids forhuman food protein composition appropriate for an adult human was FAO/WHO/UNU requirements(2007) (Table 2) [13]. Table 2. Protein of reference from FAO/WHO/UNU Essential amino acids % AA Cysteine + Methionine 1.7 Histidine 1.6 Isoleucine 1.3 Leucine 1.9 Lysine 1.6 Phenylalanine + tyrosine 1.9 Threonine 0.9 Tryptophan 0.5 Valine 1.3 Sum of EAA 12.7 AA: amino acid (g of 100 g of protein) 4 E3S Web of Conferences 183, 04004 (2020) I2CNP 2020 https://doi.org/10.1051/e3sconf/202018304004 Protein digestibility Corrected Amino Acid Score (PDCAAS) was determined according to Schaafsma [15], where it is CI × true faecal digestibility of almond meal. The true faecal digestibility in our study was used from Ahrens et al (2005) results [16]. 2.4 Statistical analysis y The chemical characterization of almond meal obtained from four varieties and one local ecotype, during three consecutive crop years, was carried out on triplicate for each sample and each parameter. Statistical analyses were conducted using Statistical Package for the Social Sciences (SPSS for Windows, version 21, SPSS Inc., Chicago, IL, USA). The normal distribution was verified according to Shapiro Wilk test. Two-way ANOVA statistical analysis and Tuckey's post-hoc test was used for means comparison, the difference was considered significant (at P<0.05). 3.1 Dry matter Dry matter data registered for almond meals (Table 3), range between a minimum of 89.29% for FF-CY1 and a maximum of 94.56 % for M-CY2. Indeed, dry matter values for analysed almond meals of each varieties are slightly different; these differences seem to be mainly related to the harvest year, the variety, and probably to the technique of post-harvest drying. The ANOVA analysis (Table 3) confirms this observation and shows a significant difference at P<0.05. In general, the results are similar to the literature, for instance, Arbouche R (2013) found 94.6% dry matter in the almond meal [17], which is very similar to our findings for M-CY2 (94.56 %). These results are comparable to those of several other types of meals (oil cakes of soybean, groundnut, coconut, cottonseed, sunflower, sesame), whose dry matter contents vary between 85% to 94% [18]. 3.3 Sugars Whatever the crop year,almond oil cake of the variety Marcona (M) had the lowest sugar content (Table 3)respectively for the three studied years (12.60%; 12.57%; 15.02%), while the variety Fournat(FNB) seems to present the highest sugar content (13.84 /21.65 % / 19.21%). The sugar contents of almond oil meals from the three other varieties are between these limit values. It is certainly here genotype-related differences, which are confirmed by ANOVA analysis (Table 4) that shows significant differences between genotypes at P<0.05. The almond oil cake, although considered simply as a by-product of almond oil extraction, is an excellent source of nutrients such as proteins, vitamins, minerals, fibres and bioactive components that may offer a number of health benefits. In this regard recently, in a research collaboration, but also from the literature [22-25], we show that high phenolic content remains in this coproduct making it usable for improvement of nutritional and functional properties of certain food products. In addition, this coproduct contains significant amounts of other phytochemicals compounds including tocopherols, flavonoids, tannins, and can be used for cosmetics and pharmaceutical purposes. In the present study, analyses focus on proteins content, amino acids profile ash and dietary fibres. 3.2 Residual oil Oil cakes/meals are solid residues, obtained after the extraction of oil from the plant part such as seeds or nuts. In general, residual oil in meals depends on quality of the raw material and on the processing of oil extraction. In spite of the fact that the process used for extraction of almond oils is the same for all varieties, the quantities of residual oil extracted from different almond meals analysed (Table 3) vary widely between a minimum of 7.45% (M -CY1) to a maximum of 23.58% (M-CY2). F-F and FNB present the highest residual oil content comparing to (M) and (B). The ANOVA analysis (Table 4) confirms this observation and shows a significant difference at P<0.001. Since the press system is the same, the difference should be related to the raw material, particularly to the physical state 5 5 E3S Web of Conferences 183, 04004 (2020) E3S Web of Conferences 183, 04004 (2020) I2CNP 2020 https://doi.org/10.1051/e3sconf/202018304004 of the kernels, moisture and kernels defects but also to the almonds variety [19]. In this regard, Rabadan et al (2017) reported comparable results that range from 15.24% and 25.5 % of residual oil in almond meals[20] and his concluded that residual oil of meals depends on efficiency of mechanical oil extraction. Besides, Savoire et al. (2013) reported that residual oil yield was influenced by many parameters such as kernels variety; press system parameters and pre-treatments preceded this process [21]. of the kernels, moisture and kernels defects but also to the almonds variety [19]. In this regard, Rabadan et al (2017) reported comparable results that range from 15.24% and 25.5 % of residual oil in almond meals[20] and his concluded that residual oil of meals depends on efficiency of mechanical oil extraction. Besides, Savoire et al. (2013) reported that residual oil yield was influenced by many parameters such as kernels variety; press system parameters and pre-treatments preceded this process [21]. 3.4 Dietary fibers and Ash Dietary fibres can act by changing the nature of the contents of the gastrointestinal tract and by changing how other nutrients and chemicals are absorbed. Fibres and minerals bring nutritional benefits to consumers. The results of the analysed almond cakes for the dietary fibres and ash contents are presented in Table 3. They vary between a minimum of 31.52% /DM registered for Marcona (M-CY2) and a maximum of 42.3%/ DM for Beldi ecotype (B-CY2). The calculated average value of dietary fibres content in analysed almond meals is 35.5%/ DM for the three crop years of this study, this value is in accordance with the literature. Analysis of ANOVA (Table 4) show no significant difference for the factor Year, whereas the factors Variety and Year Variety interaction present a significant difference between the samples at P<0.05. Finally, the fibres’ content of analysed almond meals is clearly higher than those given, by the literature, such as those of sunflower seeds (28.41%) and soybean (17.8%) [19]. 6 E3S Web of Conferences 183, 04004 (2020) I2CNP 2020 https://doi.org/10.1051/e3sconf/202018304004 E3S Web of Conferences 183, 04004 (2020) I2CNP 2020 Table 3.Summary of chemical compositions of almond mealfrom five most cultivated varieties in eastern Morocco. 7 7 3.4 Dietary fibers and Ash Results of analyses concern almond meal contents (%) for dry matter, residual oil, sugars and fibres contents, ash, proteins content and amino acid profile for three consecutive crop years (CY: Crop year; M: Marcona: FNB: Fournat e Breznaud; FF: Ferragnes Ferraduel ; B: Beldi) e naud; : e agnes e aduel ; : eldi) Sample Dry matter % Residual oil % Sugars % Dietary fibres % Ash % Proteins % M-CY1 90.44 ± 0.40ab 7.45 ± 0.20a 12.60 ± 0.64a 33.04 ± 2.43a 7.28 ± 0.00e 43.24 ± 5.38e FNB-CY1 91.51 ± 0.19b 7.66 ±0.30a 13.84 ± 0.20a 32.26 ± 2.93a 7.14 ± 0.01e 39.58 ± 0.34de F-F-CY1 89.29 ± 1.70a 8.52 ± 0.55ab 13.74 ± 0.28a 37.71 ± 3.36ab 8.03 ± 0.08fg 37.59 ± 0.34bcd B-CY1 91.16 ± 0.25b 8.07 ±0.35ab 14.20 ± 0.99a 34.86 ± 2.56ab 7.89 ± 0.10f 38.79 ± 0.21cd M-CY2 94.56 ± 1.19d 23.58 ± 1.22f 12.57 ± 0.71a 31.52 ± 5.04a 6.80 ± 0.06d 34.79 ± 2.80abc FNB-CY2 93.37 ± 0.63d 12.17 ± 0.56cd 21.65 ± 0.32d 38.42 ± 3.97ab 8.15 ± 0.11g 31.15 ± 0.13a F-F-CY2 94.06 ± 0.88d 22.48 ± 1.03e 14.48 ± 0.85ab 31.94 ± 2.93a 6.84 ± 0.03d 33.39 ± 0.01ab B-CY2 93.23 ± 0.98cd 8.71 ± 0.65ab 18.85 ± 2.90cd 42.30 ± 1.58b 7.98 ± 0.03fg 38.78 ± 0.24cd M-CY3 93.55 ± 6.44d 8.95 ± 0.84b 15.02 ± 1.30ab 35.45 ± 2.13ab 4.75 ± 0.01a 36.61 ± 1.05bcd FNB-CY3 91.80 ± 8.22bc 12.81 ± 0.38e 19.21 ± 0.55cd 39.75 ± 0.51ab 7.25 ± 0.03e 36.54 ± 0.12bcd F-F-CY3 90.74 ± 9.26ab 11.9 ± 0.11cd 15.29 ± 0.44ab 34.50 ± 0.56ab 5.88 ± 0.05b 40.18 ± 1.13de B-CY3 91.85 ± 8.15bc 10.41 ± 0.09c 17.48 ± 0.97bc 35.05 ± 2.39ab 6.14 ± 0.12c 36.50 ± 0.26bcd https://doi.org/10.1051/e3sconf/202018304004 7 https://doi.org/10.1051/e3sconf/202018304004 E3S Web of Conferences 183, 04004 (2020) E3S Web of Conferences 183, 04004 (2020) I2CNP 2020 Ash contents of the analysed almond meals from the three crop years (Table 3) varied between a minimum of 4.75 % DM for (M-CY3) and 8.15 % DM for (FNB-CY2). Analysis of ANOVA (Table 4) shows that there is a significant difference between the analysed samples at P<0.05.However, the observed differences can not be related to almond genotype or crop year effect. Almond meal is rich in macro elements, namely, potassium, phosphorus and magnesium (data not shown) which make it an important source of these essential elementsRabadan et al. (2017) [20]. In this regard, Karaman et al (2015), recommend, whenever possible, to incorporate a low dose (1 to 2%) of almond flour in food products (for instance baked goods) to improve their nutritional quality and enrich their mineral content[24]. Table 4. Variance Analyses “ANOVA two way” of constituents of almond cake/ meal, from five most cultivated varieties in eastern Morocco (NS=Non-significant, +, ++ and +++ stand for significate difference at 0.05, 0.01 and 0.001 respectively Table 4. Variance Analyses “ANOVA two way” of constituents of almond cake/ meal, from five most cultivated varieties in eastern Morocco (NS=Non-significant, +, ++ and +++ stand for significate difference at 0.05, 0.01 and 0.001 respectively Dry matter Residual oil Sugars Dietary fibres Ash Proteins Year +++ +++ +++ NS +++ +++ Variety +++ ++ +++ + +++ ++ YearVariety +++ +++ +++ ++ +++ +++ 3.5 Protein content and amino acid profiles of almond oil cake Moreover, from these results, we 8 8 E3S Web of Conferences 183, 04004 (2020) E3S Web of Conferences 183, 04004 (2020) I2CNP 2020 https://doi.org/10.1051/e3sconf/202018304004 conclude that the genotype (almond variety) strongly influences the quality (AA profiles) and the protein richness of almond seeds and consequently those of almond's oil cakes. Table 5. Amino acid profiles (Composition % of crude protein) of almond oil cake of five most cultivated varieties in eastern Morocco (M: Marcona: FNB: Fournat de Breznaud; FF: Ferragnes conclude that the genotype (almond variety) strongly influences the quality (AA profiles) and the protein richness of almond seeds and consequently those of almond's oil cakes. Table 5. Amino acid profiles (Composition % of crude protein) of almond oil cake of five most l i d i i i M (M M FNB F d B d FF F conclude that the genotype (almond variety) strongly influences the quality (AA profiles) and the protein richness of almond seeds and consequently those of almond's oil cakes. conclude that the genotype (almond variety) strongly influences the quality (AA profiles) and the protein richness of almond seeds and consequently those of almond's oil cakes. Table 5. Amino acid profiles (Composition % of crude protein) of almond oil cake of five most cultivated varieties in eastern Morocco (M: Marcona: FNB: Fournat de Breznaud; FF: Ferragnes- Ferraduel, B: Beldi).Analyses for Almonds of three consecutive crop years (CY) Table 5. Amino acid profiles (Composition % of crude protein) of almond oil cake of five most cultivated varieties in eastern Morocco (M: Marcona: FNB: Fournat de Breznaud; FF: Ferragnes- Ferraduel, B: Beldi).Analyses for Almonds of three consecutive crop years (CY) Table 5. 3.5 Protein content and amino acid profiles of almond oil cake Amino acid profiles (Composition % of crude protein) of almond oil cake of five most cultivated varieties in eastern Morocco (M: Marcona: FNB: Fournat de Breznaud; FF: Ferragnes- Ferraduel, B: Beldi).Analyses for Almonds of three consecutive crop years (CY) CY1 CY2 CY3 M FNB FF B M FNB FF B M FNB FF B Asp 4.50 4.32 4.25 4.35 5.70 4.36 4.79 5.22 4.78 4.70 5.09 5.02 Thr 1.40 1.35 1.34 1.34 1.62 1.43 1.38 1.45 1.32 1.39 1.50 1.41 Ser 1.80 1.73 1.69 1.71 2.42 1.98 2.08 2.20 1.92 1.94 2.14 1.98 Glu 8.86 8.46 8.49 8.63 13.20 9.74 11.06 11.81 10.58 10.44 11.21 10.10 Pro 1.63 1.56 1.57 1.51 2.10 1.66 1.74 1.98 1.82 1.79 2.01 1.93 Gly 2.53 2.40 2.43 2.42 3.30 2.75 2.76 2.88 2.66 2.77 2.87 2.78 Ala 1.91 1.86 1.81 1.83 2.43 1.96 1.98 2.18 1.90 1.92 2.13 1.99 Cys 0.10 0.12 0.07 0.07 0.03 - - 0.01 0.07 0.06 0.22 0.02 Val 2.48 2.38 2.35 2.39 2.07 1.73 1.75 1.93 2.10 2.18 2.42 2.30 Met 0.23 0.30 0.32 0.24 0.23 0.26 0.24 0.23 0.27 0.32 0.35 0.26 Ile 1.94 1.89 1.84 1.83 1.66 1.34 1.39 1.48 1.65 1.68 1.84 1.73 Leu 3.19 3.07 3.02 3.03 3.47 2.80 2.91 3.18 3.08 3.09 3.45 3.20 Tyr 1.47 1.39 1.37 1.38 1.40 1.14 1.16 1.29 1.30 1.26 1.46 1.34 Phe 2.45 2.37 2.30 2.31 2.56 2.04 2.14 2.27 2.29 2.30 2.47 2.28 His 1.42 1.38 1.35 1.35 1.42 1.11 1.19 1.24 1.40 1.40 1.50 1.37 Lys 1.42 1.41 1.43 1.40 1.55 1.52 1.38 1.40 1.36 1.57 1.58 1.48 Arg 4.55 4.36 4.24 4.27 5.31 3.82 4.48 4.76 4.79 4.56 5.03 4.57 From a nutritional point of view, the analysed almond meal samples show interesting protein values and a medium to pretty good amino acid profile, which is deficient in certain essential amino acids. This finding is in agreement with the results reported by Ahrens et al (2005) [16]. The mean values of chemical indices (CI) and PDCAAS (Proteins Digestibility of Corrected Amino Acid Score) are shown in Table 6. Like all vegetable proteins, which often have one or more limiting amino acids, the analysed samples of almond cakes have relatively low chemical indices (CI),which vary between a minimum of 28.22% for (M- CY2) and a maximum value of 70.81% (FF-CY3). 3.5 Protein content and amino acid profiles of almond oil cake The growing interest in almonds and derivatives is due to their nutritious ingredients such as lipids, proteins, carbohydrates, vitamins and minerals, as well as secondary metabolites. Most studies focus on almond oil, for food uses and especially in cosmetics, that is more profitable, however, few studies concern the protein fraction, which after almond oil extraction constitutes the major component of the almond cake / meal. According to US department of agriculture (USDA) database, the protein content ranges from 16.0 to 22.0% of kernel weight amongst different varieties. Therefore, theoretically after extraction of the oil, which constitutes about 50% of the weight of the kernel, protein contents must double in almond cake / meal. Indeed, protein content of the analysed almond meals from the three cropyears (Table 3) ranges between a minimum of 31.15% for (FNB-CY2) and 43.24% for (M-CY1). Similar values have been reported by Rabadan et al. (2017) [20] for almond meals of Ferragnes (40.50%) and Ferraduel (44.85%) varieties, but a high protein content (55.93%) for almond meal of Marcona, which is known for its low oil richness. ANOVA test (Table 4) shows a significant difference between the analysed almond meal samples. The significant differences observed seem to be due to both the genetic factor (almond variety) and the environment (crop year) with significant effects respectively at P<0.01 and at P<0.001, whereas the factor “Crop Year Variety” interaction present a highly significant effect at P<0.001. Amino acid (AA)profiles ofanalysed almond cake / mealarepresented in Table 5. Quantitative and qualitative analysis of AA profiles allows the identification of 17AA. The observed AA profiles of almond mealsare dominated by glutamic acid (Glu), aspartic acid (Asp), and arginine (Arg). Respectively,Glu, Asp and Arg contents (% DM) range between a minimum –maximum values of 8.46 % for (FNB-CY1) - 13.20 % for (M-CY1), 4.25 % for (FF- CY1) - 5.70 % for (M- CY2) and 3.82 % for (FNB- CY2) -5.31% for (M-CY2) (Table4). However, cysteine (Cys: 0.01 - 0.22 % DM) and methionine (Met: 0.23-0.35% DM) have the lowest contents. Regarding the essential amino acids (EAA), leucine (Leu)is the most abundant, followed by valine (Val). Finally, from a qualitative point of view, registered compositions for AA profiles of analysed almond cakes are in agreement with the finding reported by Sociasi Company et al (2008) [3]. 3.5 Protein content and amino acid profiles of almond oil cake These “CI” values are comparable to those of proteins of vegetable origin and which are generally lower than the CI of proteins of animal origin. The gap between CI values may be due to the genetic and environmental factors, which strongly influence the amino acid profiles in plants [11].All the calculated PDCAAS values for the analysed samples of almond meals are less than 100 but close to reported values for proteins of maize, rice and wheat [21]. “PDCAAS values” for almond meals range between 24.78% for (M-CY2) and 62.17% for (FF-CY3). Besides, for whole almonds,Boye et al (2012) [21], published PDCAAS values for three almond varieties (Carmel, Mission, and Nonpareil) range between 22 and 24 % and are lower than PDCCAS values for almond oil cakes of the five studied varieties (M: Marcona; FNB: Fournat de Breznaud; FF: Ferragnes-Ferraduel; B: Beldi). These differences could be explained by genetic and environmental factors. Finally, we concluded that almond meal coproducts are interesting sources of essential amino acids (EAA, Table 6). They are therefore new good sustainable nutritional alternatives to animal proteins. Table 6. 4 Conclusion This study focused on the characteristics of oil cake as an extraction residue from almond oils and showed, in view of their major constituents (sugars, proteins, fibres, and minerals), that they can be used as an ingredient or food additive, therefore, with an economic advantage for producers, but also from a zero-waste perspective of great environmental interest. Although this research was more oriented to almonds harvesting and post-harvesting processing stages, the impact of the genotype and the crop year effect, seem very visible, since the quality of the harvest almonds and their coproducts differ from year to year. This depends on the impact of agricultural conditions and interactions between almond varietiesand pedo-climatic factors. Thus, for the studied oilcake samples of different almond varieties and except their fibre contents, significant differences were observed for all the other analysed parameters (Residual oil, Dry matter, Sugars, Proteins, Ash). Although this research was more oriented to almonds harvesting and post-harvesting processing stages, the impact of the genotype and the crop year effect, seem very visible, since the quality of the harvest almonds and their coproducts differ from year to year. This depends on the impact of agricultural conditions and interactions between almond varietiesand pedo-climatic factors. Thus, for the studied oilcake samples of different almond varieties and except their fibre contents, significant differences were observed for all the other analysed parameters (Residual oil, Dry matter, Sugars, Proteins, Ash). The chemical composition, of the residues of mechanically pressed almond oil (almond meal), depends mainly on the variety of almond that, in turn, is affected by soil and climatic conditions, which determines the quality of the raw material used for the almond oil extraction. The chemical composition, of the residues of mechanically pressed almond oil (almond meal), depends mainly on the variety of almond that, in turn, is affected by soil and climatic conditions, which determines the quality of the raw material used for the almond oil extraction. Moreover, the almond meal coproducts contain beneficial nutrients and bioactive molecules; they constitute an interesting source of protein (EAA), dietary fibres and minerals; they have also the advantage to be a gluten-free product. Therefore, these co- products of almond oil extraction would be an interesting ingredient for specific gluten-free culinary preparations and in the formulation of light foodstuffs. 3.5 Protein content and amino acid profiles of almond oil cake Total essential Amino acids (ΣEAA g%); non-essential Amino acids (ΣNEAA g%); chemical index (CI), and protein digestibility corrected amino acid score (PDCAAS) 9 9 E3S Web of Conferences 183, 04004 (2020) E3S Web of Conferences 183, 04004 (2020) I2CNP 2020 https://doi.org/10.1051/e3sconf/202018304004 for almond meal coproducts of five studied almond varieties (M: Marcona; FNB: Fournat de Breznaud; FF: Ferragnes-Ferraduel, B: Beldi) ΣNEAA ΣEAA CI (Met + Cys) PDCAAS (%) (Met + Cys) M- CY1 25.76 ± 0.56 16.10 ± 0.12 47.58 ± 7.95 41.78 ± 6.98 FNB- CY1 24.69 ± 0.16 15.66 ± 0.10 60.06 ± 0.78 52.74 ± 0.68 FF- CY1 24.47 ± 0.71 15.40 ± 0.38 58.50 ± 0.25 51.37 ± 0.22 B- CY1 24.72 ± 0.13 15.34 ± 0.08 44.65 ± 1.56 39.20 ± 1.37 M- CY2 34.47 ± 0.45 16.00 ± 0.24 28.22 ± 3.59 24.78 ± 3.15 FNB- CY2 26.27 ±0.08 13.37 ± 0.08 38.42 ± 4.75 33.74 ± 4.17 FF- CY2 28.90 ± 0.19 13.53 ± 0.07 32.86 ± 1.71 28.85 ± 1.50 B- CY2 31.03 ± 0.10 14.47 ± 0.11 30.49 ± 1.12 26.77 ± 0.99 M- CY3 28.45 ± 0.17 14.82 ± 0.09 45.37 ± 1.43 39.84 ± 1.26 FN- CY3 28.13 ± 0.19 15.24 ± 0.04 51.40 ± 2.94 45.13 ± 2.58 FF- CY3 30.48 ± 0.87 16.79 ± 0.56 70.81 ± 10.12 62.17 ± 8.89 B- CY3 28.37 ± 0.24 15.39 ± 0.10 37.24 ± 0.29 32.70 ± 0.26 True faecal digestibility (%) 87.81 for almond meal coproducts of five studied almond varieties (M: Marcona; FNB: Fournat de Breznaud; FF: Ferragnes-Ferraduel, B: Beldi) 5 References 1. C. Grasselly. CIHEAM, Options Méditerranéennes., 28- 43, 32 (1976). 2. A. J. Esfahlana, R. Jameia, R. J. Esfahlan. Food. Chem.,349–360,120 (2010) 3. R. Sociasi Company, O. Kodad, J.M. Alonso and T.M. Gradziel. Almond Quality: A Breeding Perspective, in J. Janick (Eds.), Horticultural Reviews, New York, (2008). 4. A.Elamrani, R.Melhaoui, N.Houmy, M. Addi, M.Abid, A.Mihamou, M.L.Fauconnier, M.Sindic& H.Serghini-Caid. Valorisation Challenges to Almonds and their Co- products: Characterization of Oils Extracted from Broken Almonds Springing from Shelling and Sorting Operations.In proceeding of the fourth American Moroccan Agricultural Sciences Conference, 9-11May 2018, Meknes, Morocco (2018). 5. ISO 662. International Organisation of Standardisation. Corps gras d'origines animale et végétale — Détermination de la teneur en eau et en matières volatiles (1998) 6. C.S. James. Analytical Chemistry of Foods: Determination of fat by the Soxhlet methods. Blackie Academic and Professional, ed., C.S. James, London, (1995). ( ) 7. State standard of Russian Federation GOST5672-68. Method for determining the mass fraction of sugar. Bread and bakery products. (1968) 8. A.O.A.C. 991. 43. Association of Official Analytical Chemists: Total, Soluble, and Insoluble Dietary Fibre in Foods: Enzymatic-Gravimetric Method, Mes-Tris Buffer (1996). ( ) 9. A.A.C.C. 08-01. American Association of Cereal Chemists. Ash: Basic method (2001). 10 A F NOR NF V04-407 L’Association Française de Normalisation Détermination de la 9. A.A.C.C. 08-01. American Association of Cereal Chemists. Ash: Basic method (2001). 9. A.A.C.C. 08-01. American Association of Cereal Chemists. Ash: Basic method (2001). 10. A.F.NOR. NF.V04-407. L’Association Française de Normalisation. Détermination de la teneur en azote total et calcul de la teneur en protéines – Méthode Kjeldahl (2002). Á 10. A.F.NOR. NF.V04-407. L’Association Française de Normalisation. Détermination de la teneur en azote total et calcul de la teneur en protéines – Méthode Kjeldahl (2002). 12. D. H. Spackman, W. H. Stein, and Stanford. Anal. Chem., 1190–1206,30, 7 (1958). 13. FAO/WHO/ONU. Food and Agriculture Organisation/ World Health Organization/ Organization of United Nations. Protein and amino acid requirements in human nutrition. Technical Report Series 935. Geneva, Switzerland. (2007). 14. A. Sujak, A. Kotlarz, W. Strobel. Food Chem., 711-719,98 (2006) 15. G. SchaafsmaThe Protein Digestibility–Corrected Amino Acid Score, in Proceedings of the symposium Criteria and Significance of Dietary Protein Sources in Humans, 4 October 1999, San Francisco, USA (1999). 16. S. Ahrens, M. Venkatachalam, A. M. Mistry, K. Lapsley& S. K. Sathe. Plant. Food. Hum. Nutr., 123–128,60 (2005). 17. R. 4 Conclusion Indeed, world protein demand is growing strongly and it seems necessary to offer new sustainable and good nutritional alternatives to animal protein. Legume cakes, such as soybean and rapeseed meals, because of their relatively balanced essential amino acid profile (EAA), are widely used in the formulation of animal feed and vegan food. Almond oil cakes, despite their relatively unbalanced essential amino acid (EAA) profile, have the advantage of being 10 E3S Web of Conferences 183, 04004 (2020) E3S Web of Conferences 183, 04004 (2020) I2CNP 2020 https://doi.org/10.1051/e3sconf/202018304004 gluten-free and could be a new ingredient for the formulation of new light foodstuffs and vegan foods. gluten-free and could be a new ingredient for the formulation of new light foodstuffs and vegan foods. Acknowledgements -We are grateful for the Moroccan Higher Education Ministry and Wallonia Brussels International (WBI Project 2-4) for supporting this research.The assistance of PRODIGIA Company for the mechanical extraction of almond oils and the almond growers in eastern morocco for providing the almond samples for this study. 5 References Arbouche,Incorporation du tourteau d’amande d’abricot en substitution au tourteau de soja dans l’alimentation des animaux domestiques: Ruminants (cas des ovins à l’engrais) et des monogastriques (cas du poulet de chair).Doctoral Thesis,ElHadj Lakhdar University, Batna, Algeria (2013). 18. S. Ramachandran, S. K. Singh, C. Larroche,C. R. Soccol, A. Pande. Bioresource. Technol.,2000–2009, 98 (2007) 19. S. Sivaramakrishnan& D. Gangadharan. Chapter 13: Edible Oil Cakes. Biotechnology Division, National Institute for Interdisciplinary Science and Technology (NIIST), CSIR, Trivandrum 695 019, Kerala, India (2009). 11 E3S Web of Conferences 183, 04004 (2020) I2CNP 2020 https://doi.org/10.1051/e3sconf/202018304004 20. A. Rabadán, M. Álvarez-Ortí, R. Gómez, A. Pardo-Giménez, J. E. Pardo. Sci. Hortic. Amsterdam., 539–546,225 (2017). ( ) 21. R. Savoire, J.L. Lanoisellé, E. Vorobiev. Food. Bioprocess. Tech., 1–16,6 (2013) 22. D.Tungmunnithum, A. Elamrani, M. Abid, S.Drouet R.Kiani, L.Garros, A.Kabra, M.Addi and C. Hano.Quick, Green and Simple Ultrasound-Assisted Extraction for the Valorization of Antioxidant Phenolic Acids from Moroccan Almond Cold-Pressed Oil Residues, Appl. Sci. 10 (2020). 23. D.Tungmunnithum, M. Abid, A.Elamrani, S.Drouet, M.Addi and C.Hano. Life.10 (2020). 24. S. Karaman, S. Karasu,F.Tornuk, O.Toker, U.Geçgel, O. Sagdic. J.Agr. Food. Chem., 2305–2313,63(2015). Č 25. S. Čolić, G. Zec, M.Natić, and M.Fotirić-Akšić. Almond (Prunus dulcis) oil, in e-book “Fruit Oils: Chemistry and Functionality”. M. F. Ramadan Editor (Springer Nature Switzerland, AG 2019). 26. J. Boye, R. Wijesinha-Bettoni and B. Burlingame. Brit. J. Nutr., S183–S211, 108 (2012). 12 12
https://openalex.org/W4385856920	http://repositorio.udec.cl/jspui/bitstream/11594/10808/1/Sehlke%20Abarca%2c%20Kirsty%20Tesis.pdf	es		Formación de Agujeros Negros a través de colisiones de estrellas de la secuencia principal en cúmulos primordiales con un potencial de fondo (formation of black holes via collisions of main-sequence stars in primordial clusters with a background potential).	null	2,023	cc-by	21,447	UNIVERSIDAD DE CONCEPCIÓN FACULTAD DE CIENCIAS FÍSICAS Y MATEMÁTICAS FORMACIÓN DE AGUJEROS NEGROS A TRAVÉS DE COLISIONES DE ESTRELLAS DE LA SECUENCIA PRINCIPAL EN CÚMULOS PRIMORDIALES CON UN POTENCIAL DE FONDO (FORMATION OF BLACK HOLES VIA COLLISIONS OF MAIN-SEQUENCE STARS IN PRIMORDIAL CLUSTERS WITH A BACKGROUND POTENTIAL) Por: Kirsty Sehlke Abarca Tesis presentada a la facultad de ciencias físicas y matemáticas de la Universidad de Concepción para optar al grado académico de Magíster en Ciencias con Mención en Física. Abril 2023 Concepción, Chile Profesor Guía: Dominik Schleicher - UdeC Dedicada a mi madre Mariana Abarca Oyarzún i AGRADECIMIENTOS Primero quiero comenzar agradeciendo a mi hermosa madre Mariana Abarca Oyarzún quien fue la persona que sembró un montón de semillitas en mi y las fue regando por mi hasta que yo pude regarlas por mi misma. Ella me mostró su amor por las matemáticas siendo una excelente Ingeniera de ejecución en mecánica y fue con su ejemplo que me he convertido en la mujer que soy hoy en día. Ella fue la que seco mis lágrimas cada vez que quise rendirme, aplaudió y se emociono conmigo en cada uno de mis triunfos y fue, es y seguirá siendo hasta mi ultimo día el motor que impulsa mis sueños. Gracias mama porque me mostraste que nada de lo que uno quiera alcanzar es imposible si te propones obtenerlo. Gracias porque aun yo sin creer en mi tu si creías en mi y a través de tus ojos yo podía verlo. Yo se que tu estabas muy orgullosa de mi y de ver todo ese camino que he recorrido de tu mano. Ahora tu sueltas mi mano físicamente y me dejas volar sola poniendo en practica cada una de las enseñanzas que me dejaste para el presente y el futuro pero no dudo que me seguirás apoyando desde el cielo. Gracias por ser mi madre, mi compañera, mi guía y por mostrarme como se siente ser amada incondicionalmente y haberme dado el honor de acompañarte en los últimos años de tu vida que se no fue fácil. Te extraño muchísimo y como te dije una de las ultimas veces que hablamos: "Mujeres fuertes inspiran niñas fuertes" y eso fuiste para mi la mujer mas fuerte que he conocido. Te amo mama. Ella con el dolor de mi alma falleció (21 de mayo de 1951 - 4 de noviembre del 2021) durante mi segundo año de magíster y porque se que así ella lo hubiese querido termine este magíster por ella. Quiero también agradecer a mis tres mejores amigas de hace casi una década que han estado conmigo incondicionalmente que ahora son mi familia en especial en este ultimo tiempo que ha sido tan difícil para mi. Agradecer a mis compañeros y compañeras de universidad con los cuales pude trabajar a la par independientemente de ser mujer en un campo mayoritariamente compuestos por hombres y por esas largas noches de estudios y cervezas que las hicieron mucho mas entretenidas con su compañia. Por otro lado quiero agradecer a mi profesor guía Dominik Schleicher por dejarme formar parte de este grupo de agujeros negros, por ser un excelente profesor guía ii y por el apoyo y comprensión que me dio en el que seguro va a ser el momento mas difícil de mi vida. También quiero agradecer a mi otro profesor guía Andrés Escala por siempre estar presente para explicar y guiar de muy buena manera mi investigación en complemento de la suya. Finalmente agradecerle a la Universidad de Concepción por la beca que obtuve para realizar mi magíster en conjunto con mi titulo de pregrado a través de la beca de articulación y al soporte financiero a ANID con el proyecto Millenium Nucleus NCN19_058 (TITANs), que permite explorar de diversas formas los agujeros negros supermasivos desde su formación, crecimiento, características y tecnologías, por la beca que obtuve durante todo el proceso que duro el trabajo de esta investigación que se presenta a continuación. iii Resumen La asombrosa existencia de agujeros negros supermasivos en el Universo primitivo es uno de los grandes misterios sin respuesta en astrofísica, en particular, cómo obtienen sus grandes masas tan rápido. Aquí nuestro interés consiste en explorar un nuevo escenario de formación de agujeros negros masivos: la formación por colisiones estelares catastróficas en cúmulos estelares densos. Nuestros sistemas están localizados en el universo temprano, con las denominadas estrellas de población III las cuales son muy masivas y, por tanto, cúmulos de estrellas de población III son sistemas estelares muy densos y masivos. Las simulaciones de N cuerpos fueron corridas con el código NBODY6++GPU y pueden ser divididas en dos grupos, uno con potencial de fondo y otro sin potencial de fondo. Se ha establecido una masa crítica para la cual el número de colisiones es tan alto que puede llegar a formar agujeros negros masivos al interior de estos cúmulos estelares nucleares. Aquí, nuestro objetivo es testear la existencia de esta masa crítica en un caso simplificado de cúmulos de estrellas de población III de igual masa. En términos de la eficiencia para la formación de un objeto masivo, encontramos que ocurre una transición, ya que la eficiencia es muy pequeña para masas considerablemente por debajo del valor de masa crítica, alcanzando valores de ∼ 20% una vez que comenzamos a acercarnos a la escala de masa crítica en nuestras simulaciones. Nuestros resultados sugieren que ocurre una transición crítica dependiendo de la masa del cúmulo y los objetos más masivos alcanzan masas de aproximadamente 1.7 × 105 M. Keywords – agujeros negros: formación – universo temprano – cúmulos estelares densos – colisiones estelares catastróficas – simulaciones iv Abstract The amazing existence of supermassive black holes in the primitive Universe is one of the great mysteries without answer in astrophysics, in particular, how they get their great masses so fast. Here our interest consists in exploring a new scenario for the formation of massive black holes: the formation by catastrophic stellar collisions in dense star clusters. Our systems are located in the early universe, with the so-called population III stars which are very massive, and therefore population III star clusters are very dense and massive stellar systems. The N-body simulations we run with the code NBODY6++GPU can be divided into two groups, one with background potential and the other without background potential. A critical mass has been established for which the number of collisions is so high that massive black holes can form within these nuclear star clusters. Here, our goal is to test the existence of this critical mass in a simplified case of population III star clusters of equal mass. In terms of the efficiency for the formation of a massive object, we find a transition to occur, as the efficiency is very small for masses considerably below the critical value while reaching values of ∼ 20% once we start approaching the critical mass scale in our simulations. Our results thus suggest such a critical transition to occur depending on the mass of the cluster and the most massive objects reaching masses of about 1.7 × 105 M. Keywords – black hole: formation – early universe – dense stellar clusters – catastrophic stellar collsions – simulations Contents v Contents AGRADECIMIENTOS i Resumen iii Abstract iv 1 Introduction 1.1 Supermassive black holes in the early universe . . . . . . . . . . . 1.2 Poulation III stars . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Nuclear star clusters . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Previous work on clusters and collisions of Population III stars . . 1.5 Comparison with theoretical expectations for nuclear clusters at galaxy centers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 5 7 10 12 2 Methodology 17 2.1 NBODY6++ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 2.1.1 Hermite scheme . . . . . . . . . . . . . . . . . . . . . . . . 17 2.1.2 Individual and block time steps . . . . . . . . . . . . . . . 19 2.1.3 Ahmad–Cohen scheme . . . . . . . . . . . . . . . . . . . . 20 2.1.4 Regularization of close encounters and few-body subsystems 23 2.2 Description of the numerical setup . . . . . . . . . . . . . . . . . . 28 2.2.1 Input . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 2.2.2 Initial conditions . . . . . . . . . . . . . . . . . . . . . . . 31 2.3 Collision treatment . . . . . . . . . . . . . . . . . . . . . . . . . . 32 3 Analysis 3.1 Pop. III star cluster simulations . . . . . . . . . . . . . . . . . . . 3.2 Theoretical results for Pop. III star clusters . . . . . . . . . . . . 3.3 Efficiencies for Pop. III star clusters . . . . . . . . . . . . . . . . . 3.4 Summary of the main results . . . . . . . . . . . . . . . . . . . . . 34 34 40 43 48 4 Discussion 51 5 Conclusion 54 Referencias 57 vi List of Tables List of Tables 2.2.1 Initial conditions of the simulations. . . . . . . . . . . . . . . . . . 32 3.4.1 Main results from the simulations at 1 Myr. . . . . . . . . . . . . 3.4.2 Main results from the simulations at 5 Myr. . . . . . . . . . . . . 3.4.3 Main results from the simulations at 10 Myr. . . . . . . . . . . . . 48 49 50 List of Figures vii List of Figures 1.1.1 A representation of the evolution of the universe over 13.77 billion years. Source: (NASA / WMAP Science Team, 2006). See the page here https://map.gsfc.nasa.gov/media/060915/index.html. . 1.1.2 MBH formation scheme. Source: (Begelman and Rees, 2009). . . . 1.3.1 Flow chart of an isolated halo with Z > Zcrit , starting from gas cooling until the formation of an NSC in the center of the halo. Source: (Lupi et al., 2014). . . . . . . . . . . . . . . . . . . . . . . 1.3.2 Flow chart reporting the NSC contraction process leading to the formation of a single seed BH from the merger among stellar mass black holes. Source: (Lupi et al., 2014). . . . . . . . . . . . . . . . 1.4.1 Evolution of a cluster with N = 1000 stars, total mass Mstars = 104 M , Rv = 0.14 pc, and Rstar = 100 R . Source: (Reinoso et al., 2020). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.2 Evolution of a cluster with N = 1000 stars, total mass Mstars = 104 M , Rv = 0.14 pc, and Rstar = 100 R and in the center of the cluster an external potential with Mext = 104 M . Source: (Reinoso et al., 2020). . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.1 Measured masses versus effective radius diagram. From: (Escala, 2021). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.2 Mass of the CMO versus the observed efficiency of the black holes formation diagram. Source: (Escala, 2021). . . . . . . . . . . . . . 1.5.3 Collision and relaxation timescales in the mass versus radius diagram. Source: (Escala, 2021). . . . . . . . . . . . . . . . . . . 2.1.1 Illustration of the block time step scheme in NBODY6++ GPU for four particles. Source: NBODY6++ GPU Manual for the computer code (Khalisi et al., 2019). You can find this document here https://github.com/nbodyx/Nbody6ppGPU/tree/master/doc. 2.1.2 A neighbor scheme for a particle marked as the asterisk. Source: NBODY6++ GPU Manual for the computer code (Khalisi et al., 2019). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3 Illustration of the regular and irregular time steps. Source: NBODY6++ GPU Manual for the computer code (Khalisi et al., 2019). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 4 8 9 11 12 14 15 16 20 22 23 viii List of Figures 2.1.4 Flow chart NBODY6++ GPU. Source: NBODY6++ GPU Manual for the computer code (Khalisi et al., 2019). . . . . . . . . . . . . 2.1.5 Flow chart NBODY6++ GPU. Source: NBODY6++ GPU Manual for the computer code (Khalisi et al., 2019). . . . . . . . . . . . . 3.1.1 Evolution of a star cluster with 104 stars with an external potential of the same mass. Each star in the cluster has the same mass and radius of 30 M and 2.12 R , respectively. The virial radius is 0.05 pc and the cluster has evolved to 10 Myr. The first panel shows the number of collisions over time, the second panel shows the evolution of the Lagrangian radii of the cluster at 10%, 50% and 90% of the enclosed mass, the third panel shows the mass of the most massive object in the cluster divided by the initial mass of each star and the last panel shows the number of escaping stars divided by the total number of stars. Source: Kirsty L.K. Sehlke-Abarca et. al. in preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2 Evolution of a star cluster of 104 stars without an external potential, each star in the cluster has the same mass and radius 30 M and 2.12 R , respectively. The virial radius is 0.05 pc and the cluster has evolved to 10 Myr. The first panel shows the number of collisions over time, the second panel shows the evolution of the Lagrangian radii of the cluster at 10%, 50% and 90% of the mass, the third panel shows the mass of the most massive object in the cluster divided by the initial mass of each star and the last panel shows the number of escaping stars divided by the total number of stars. Source: Kirsty L.K. Sehlke-Abarca et. al. in preparation. . . . . . 3.2.1 In this figure the radius of the cluster in pc is shown on the x-axis while on the y-axis is the mass of the cluster in M . The different colors in the figure represent the different initial stellar masses for both triangles and lines. In this case, yellow represents the initial mass of each star of 15 M , cyan represents the initial mass of each star of 30 M , magenta represents an initial mass of each star of 50 M and finally coral represents an initial mass of each star of 100 M . Triangles pointing to the right (B) represent simulations with an external potential on the other hand triangles pointing to the left (C) represent simulations without an external potential. The solid lines are from the condition of equation 3.2.1 for the different σ associated with the initial conditions, while the dashed lines are from equation 3.2.2 both for a time evolution of 1 Myr. Source: Kirsty L.K. Sehlke-Abarca et. al. in preparation. . . . . . . . . . 26 27 36 39 41 List of Figures 3.2.2 In this figure the radius of the cluster in pc is shown on the x-axis while on the y-axis is shown the mass of the cluster in M . The different colors in the figure represent the different initial stellar masses for both triangles and lines, in this case, yellow represents an initial mass of each star of 15 M , cyan represents an initial mass of each star of 30 M , magenta represents an initial mass of each star of 50 M and finally coral represents an initial mass of each star of 100 M . Triangles pointing to the right (B) represent simulations with an external potential on the other hand triangles pointing to the left (C) represent simulations without an external potential. The solid lines are from the condition of equation 3.2.1 for the different σ associated with the initial conditions, while the dashed lines are from equation 3.2.2 both for a time evolution of 5 Myr. Source: Kirsty L.K. Sehlke-Abarca et. al. in preparation. . . 3.2.3 In this figure the radius of the cluster in pc is shown on the x-axis while on the y-axis is shown the mass of the cluster in M . The different colors in the figure represent the different initial stellar masses for both triangles and lines, in this case, yellow represents an initial mass of each star of 15 M , cyan represents an initial mass of each star of 30 M , magenta represents an initial mass of each star of 50 M and finally coral represents an initial mass of each star of 100 M . Triangles pointing to the right (B) represent simulations with an external potential on the other hand triangles pointing to the left (C) represent simulations without an external potential. The solid lines are from the condition of equation 3.2.1 for the different σ associated with the initial conditions, while the dashed lines are from equation 3.2.2 both for a time evolution of 10 Myr. Source: Kirsty L.K. Sehlke-Abarca et. al. in preparation. 3.3.1 Efficiencies at which MMOs are formed calculated with equation 3.3.3. The x-axis shows the initial stellar mass of the cluster Mini divided by the critical mass Mcrit until 1 Myr. Source: Kirsty L.K. Sehlke-Abarca et. al. in preparation. . . . . . . . . . . . . . . . . 3.3.2 Efficiencies at which MMOs are formed calculated with equation 3.3.3. The x-axis shows the initial stellar mass of the cluster Mini divided by the critical mass Mcrit until 5 Myr. Source: Kirsty L.K. Sehlke-Abarca et. al. in preparation. . . . . . . . . . . . . . . . . 3.3.3 Efficiencies at which MMOs are formed calculated with equation 3.3.3. The x-axis shows the initial stellar mass of the cluster Mini divided by the critical mass Mcrit until 10 Myr. Source: Kirsty L.K. Sehlke-Abarca et. al. in preparation. . . . . . . . . . . . . . . . . ix 42 43 44 45 46 Chapter 1. Introduction 1 Chapter 1 Introduction 1.1 Supermassive black holes in the early universe One of the most important questions in this field is: How did the first supermassive black holes (SMBHs) grow so large so fast? The answer is that the dominant formation pathway of SMBHs is still a mystery. The presence of SMBHs with masses greater than 109 M when the Universe was a billion years old represents an intriguing puzzle. Some of the theories to answer this question are that very massive black hole seeds were formed, and that black holes (BHs) have grown extremely rapidly either via accretion of the surrounding gas at super Eddington rates or collisions with other smaller BHs. How are BHs thought to have formed? So far there are three theories of how these massive black holes (MBHs) formed. The first is the direct collapse of a primordial cloud, in which primordial clouds of interstellar gas collapse under self-gravity to form supermassive stars which then evolve into supermassive black holes. The second is growth by gas accretion. And the third is the formation of an MBH by catastrophic stellar collisions in dense stellar clusters. A naive explanation (to the question we asked ourselves earlier) is that these early SMBHs were seeded by BH remnants of the first Population III stars. Pop III stars are expected to form in dark matter minihalos from primordial gas undergoing molecular hydrogen cooling. The metal-free primordial gas is significantly warmer (a few 100 K) than star-forming molecular clouds in the interstellar medium (ISM) in our Galaxy (≈10 K). The general expectation is that inefficient cooling of the primordial gas leads to inefficient fragmentation, making Pop III stars unusually 2 1.1. Supermassive black holes in the early universe massive (Inayoshi et al., 2020). Population III stars formed approximately 400 million years after the Big Bang as shown in the figure 1.1.1. Figure 1.1.1: A representation of the evolution of the universe over 13.77 billion years. Source: (NASA / WMAP Science Team, 2006). See the page here https://map.gsfc.nasa.gov/media/060915/index.html. The left side in figure 1.1.1 represents the earliest time in the universe that we can now probe when a period of "inflation" produced a burst of exponential growth in the universe. (The size is represented by the vertical extent of the grid in this graphic.) Over the next few billion years, the expansion of the universe gradually slowed down as the matter in the universe attracted itself through gravity. In recent times, the expansion began to speed up again as the repulsive effects of dark energy started to dominate the expansion of the universe. In the figure, you can also see the Wilkinson Microwave Anisotropy Probe (WMAP), a NASA spacecraft operating from 2001 to 2010 which measured the temperature differences across the sky in the cosmic microwave background (CMB), the radiant heat remaining from the Big Bang. The residual light seen by WMAP was emitted about 375,000 years after inflation and has traversed the universe largely unhindered ever since. The conditions of previous times are imprinted in this light and it is in this way that we can study it; it also forms a backlight for later developments in the universe. In figure 1.1.2 you can see the different possible paths of black hole formation that are studied today. Each with different objects and under different conditions. To explain the formation of supermassive black holes, intermediate-mass black holes (IMBHs) have also been proposed, where different candidates have been 1.1. Supermassive black holes in the early universe 3 identified as summarized by (Greene et al., 2020), but none have been confirmed and from both theoretical and observational perspectives, gravitational runaway is unlikely to take off in typical globular clusters, though the most massive star clusters may still host such events. Finally, interesting works have been carried out that can be read for more detail in (Kashlinsky, 2021) about cosmological advection flows in the presence of primordial black holes as dark matter in the context of the formation of first sources of light. 4 1.1. Supermassive black holes in the early universe Figure 1.1.2: MBH formation scheme. Source: (Begelman and Rees, 2009). 1.2. Poulation III stars 1.2 5 Poulation III stars The objects of this era marked the end of the cosmic dark age and the first stars in the universe arose a few hundred million years after the Big Bang, leading to a fundamental transformation of the early Universe and the initial enrichment with heavy chemical elements. In the early Universe, the primordial gas from which the first stars formed was composed mainly of hydrogen, helium, and metal-free. It is still not known exactly how these stars were formed and the real values of the physical characteristics of these stars since we cannot see them directly because they are too faint to be directly detectable at high redshift, that is why we can only study them through numerical methods in simulations with supercomputers. The temperature of the gas at that time was very high because the cooling was less efficient than in the present-day Universe which has more metals and dust. It has been studied the generic spectral signature of an early population of massive stars at high redshifts from which it has been obtained that for metal-free stars the generic spectrum resembles a black body with an effective temperature of 105 K, making these stars highly efficient at ionizing hydrogen and helium (Bromm et al., 2001). Supermassive Population III stars ' 104 M have also been studied to explain the formation of the first quasars (Haemmerlé et al., 2018). A large number of scientists agree that the mass of these stars varies between 10 M and 100 M . Since these stars are massive they live less than stars like the sun. Variations of the initial mass function and also a universal mass function for these stars have been studied by (Kroupa, 2001). The first stars are thought to be one of the dominant sources of hydrogen reionization in the early Universe, with their high luminosities and surface temperatures expected to drive high ionizing photon production rates (Murphy et al., 2021). Because they are poor in metals these production rates are higher (Schaerer, 2002). Theoretical studies have been done with the use of the Λ Cold Dark Matter (ΛCDM) model, which explains the observations of the cosmological structure since it provides the initial conditions for the primordial star formation (Bromm, 2013a). From this time to the present the universe has expanded rapidly and has also cooled. Studies that include the effect of gas cooling via molecular hydrogen suggest that the minihalos have a total mass of ≈ 106 M and collapsed at redshifts z ' 20-30 (Bromm, 2013b). Some of the latest studies that have been 6 1.2. Poulation III stars done related to predictions of what the James Webb Space Telescope might observe of these stars see (Windhorst et al., 2018), a satellite that was finally launched a few months ago on December 25, 2021. It is also a prediction that ELT-HARMONI will be able to observe population III stars (Grisdale et al., 2021). Population III stars were called the stars of the early universe that formed from the primordial gas, being these very massive and with zero metallicity. Since these stars are at high redshift, they are too weak to be studied observationally. This is why what we know is based on theoretical studies accompanied by simulations. At first, it was thought that this could have been a not-so-complicated process since the environment was mostly composed of hydrogen and helium, but today we know that this process is as complicated as the current star formation process. Now we know that the accretion disks that build up around the first stars are highly susceptible to fragmentation. So it has been proposed that Population III stars are binaries or members of multiple stellar systems and clusters. It is estimated that the masses of these stars are very wide. The masses have a relatively flat distribution that spans the substellar regime up to several hundred solar masses, with the most likely values being around a few tens of solar masses (Klessen, 2019). In current numerical simulations of the star formation process, more knowledge is lacking about the effect of higher resolution in the simulations, a larger fraction of the star formation timescales, including important physical processes like the protostellar feedback, magnetic fields, heating from dark matter annihilation, WIMP dark matter, cosmic rays, relative streaming velocity between baryons and dark matter in the center of a halo or a three-dimensional approach to understanding more about the gas fragmentation or the dynamical impact of stellar radiation on the infalling gas. Therefore there is still a large computational gap to resolve to understand the star formation process. This area of study is relatively new since it has only been possible to study it with the development of new numerical methods and very powerful supercomputers. There are still no simulations that include all the physical processes involved in a single simulation since it is very complex. At that time, H2 was formed more efficiently as H + e − → γ + H− H− + H → H2 + e− 1.3. Nuclear star clusters 7 (the H− channel) via gas-phase reactions in a cloud of primordial composition. Future missions such as Transient High-Energy Sky and Early Universe Surveyor (THESEUS), a space telescope that will observe Population III stars, will help to understand among other physical processes the life cycle of the first stars and thus the cosmic history of star formation (Tanvir et al., 2021). 1.3 Nuclear star clusters Nuclear star clusters (NSCs) are dense and massive assemblies of stars found at the centers of most galaxies. Many properties of NSCs vary with the properties of their host galaxies. It has also been studied that a clear transition occurs in galaxies with a mass of ≈ 109 M where the characteristics of nuclear star clusters change. The latest work on the formation of star clusters shows that atomic-cooling halos in which the star clusters form are affected by dark matter motions which reduce the stellar tidal disruption events rates (Sakurai et al., 2019). A possible route of black hole seed formation has been explored that appeals to a model by Davies, Miller & Bellovary who considered the case of the dynamical collapse of a dense nuclear cluster of stellar black holes subjected to an inflow of gas at the center of pregalactic discs forming at very high redshift. They found that this route is feasible, the formation peaks at redshifts z / 10 and occur in concomitance with the formation of seeds from other channels. The channel is competitive relative to others and is independent of the metal content of the parent cluster. This mechanism of gas-driven core collapse requires inflows with masses at least 10 times larger than the mass of the parent star cluster, occurring on time scales shorter than the evaporation/ejection time of the stellar black holes from the core. In this respect, the results provide an upper limit to the frequency of this process (Lupi et al., 2014). Figure 1.3.1 and figure 1.3.2 show a schematic of the formation of nuclear star clusters and black hole seeds from these nuclear star clusters. 8 1.3. Nuclear star clusters Figure 1.3.1: Flow chart of an isolated halo with Z > Zcrit , starting from gas cooling until the formation of an NSC in the center of the halo. Source: (Lupi et al., 2014). 1.3. Nuclear star clusters 9 Figure 1.3.2: Flow chart reporting the NSC contraction process leading to the formation of a single seed BH from the merger among stellar mass black holes. Source: (Lupi et al., 2014). Other important references are (Regan and Haehnelt, 2009) who studied pathways to form massive black holes and compact star clusters in pre-galactic dark matter haloes with virial temperatures ' 10 000 K where black holes form at the centers via a stellar seed black hole, a quasistar or via a nuclear star cluster in dark matter (DM) haloes. The latest studies of runaway collisions in dense clusters that may lead to the formation of supermassive black hole (SMBH) seeds found that mass loss can significantly affect the final mass of the possible SMBH seed. Considering a constant mass loss of 5 % for every collision, it can lose between 60-80 % of the total mass that is obtained if the mass loss were not considered. Using instead analytical prescriptions for the mass loss, the mass of the final object is reduced by 15-40 %, depending on the accretion model for the cluster that is studied (Alister Seguel et al., 2020). It has also been studied how supermassive stars (SMSs) with masses ≈ 103 − 105 M could be formed via gas accretion and runaway stellar collisions in high-redshift, metal-poor nuclear star clusters (NSCs) and after the formation of SMBHs through the collapse of supermassive stars (SMSs) into seed black holes which could grow up to the SMBHs with a few times 109 M observed at z ≈ 7. These are highly sensitive to the initial conditions and the assumed recipe for the accretion, due to the highly chaotic nature of the problem (Das et al., 2021). 10 1.4. Previous work on clusters and collisions of Population III stars Another topic of investigation is the interplay among stellar dynamics, gas accretion, and protostellar evolution. Gas accretion onto the protostars enhances their radii, resulting in an enhanced collisional cross-section. The fraction of collisions can increase from 0.1 to 1 % of the initial population to about 10 % when compared to gas-free models or models of protostellar clusters in the local Universe. This way very massive objects can form despite initial fragmentation, making the first massive protostellar clusters viable candidate birthplaces for observed supermassive black holes (Boekholt et al., 2018). For clusters with a moderate initial central concentration and a realistic IMF, the ratio of core-collapse time to initial half-mass relaxation time is typical ≈ 0.1, in agreement with the value previously found by direct N-body simulations for much smaller systems. Remarkably, for all realistic initial conditions, the mass of the collapsing core is always close to ≈ 10−3 of the total cluster mass, very similar to the observed correlation between the central black hole mass and the total cluster mass in a variety of environments (Gurkan et al., 2004). 1.4 Previous work on clusters and collisions of Population III stars One of the latest works that have been pursued is that of (Reinoso et al., 2018) who concluded that collisions in massive Pop. III clusters were likely relevant to form the first intermediate-mass black holes, particularly in more massive Pop. III clusters as expected in the first atomic cooling halos. They derived a more significant enhancement by a factor of 15 and 32 for such massive systems. On the other hand, it has been found by (Reinoso et al., 2020), who included a background potential and compared to the results of simulations with no background potential with simulations that did have a background potential, that the background potential increases the velocities of the stars, causing an overall delay in the evolution of the clusters and the runaway growth of a massive star at the center. In this case, the population of binary stars is lower due to the increased kinetic energy of the stars, initially reducing the number of stellar collisions, and the relaxation processes are also affected. On the other hand, the external potential enhances the mass of the merger product by a factor ≈ 2 if the collisions are maintained for a long time. 1.4. Previous work on clusters and collisions of Population III stars 11 Figure 1.4.1: Evolution of a cluster with N = 1000 stars, total mass Mstars = 104 M , Rv = 0.14 pc, and Rstar = 100 R . Source: (Reinoso et al., 2020). In figures 1.4.1 and 1.4.2 the uppermost panel shows the number of mergers as a function of time. The second panel shows the fraction of binaries, hyperbolics (which are stars that are not gravitationally bound), chains (which occur between stars that are part of a higher-order system), and the stars that escape from the cluster as a function of time. The third panel shows the 10 %, 50 %, and 90 % Lagrangian radii as a function of time. And the final panel shows the mass growth of the most massive object in the cluster divided by the initial mass of the stars as a function of time. Here we can see how the collisions occur afterward when we simulate the effects of the gas with an external potential. The number of stars that escape from the cluster is much smaller in the presence of an external potential, the central part of the cluster that we can see if we enclose 10% of the mass of the cluster collapses while the outermost parts expand and finally with an external potential the mass of the central object becomes larger in time. 1.5. Comparison with theoretical expectations for nuclear clusters at galaxy 12 centers Figure 1.4.2: Evolution of a cluster with N = 1000 stars, total mass Mstars = 104 M , Rv = 0.14 pc, and Rstar = 100 R and in the center of the cluster an external potential with Mext = 104 M . Source: (Reinoso et al., 2020). 1.5 Comparison with theoretical expectations for nuclear clusters at galaxy centers In the paper (Escala, 2021) the author proposes a new formation scenario for massive black hole formation driven by stellar collisions in galactic nuclei in which massive black holes and nuclear stellar clusters are different evolutionary paths of a common formation mechanism. The collision time scale for any virialized stellar system is defined by 1 tcoll = nΣ0 r R , GM (1.5.1) where n is the number density, Σ0 is the effective cross section, R is the characteristic radius of the system, G is the gravitational constant and M is the total mass of the cluster. If the age of the universe, tH , is comparable or longer than the collision time tcoll 1.5. Comparison with theoretical expectations for nuclear clusters at galaxy centers 13 ≤ tH , it is equivalent to the following condition 4η 2 300R tH G1/2 2/3 ≤ M R−7/3 , (1.5.2) where η is the mass of the stars normalized by M . And the other constants M , R , tH and G are the solar mass, the solar radius, the age of the universe and the gravitational constant, respectively. If we consider that a virialized system has a radius R equal to R = GM/σ 2 , equation 1.5.2 can be written as s 4η η ≤ 300σ tH M σ σ 3.5 , (1.5.3) where σ is the characteristic dispersion velocity of the system and σ is the solar characteristic dispersion velocity of the system. If the age of the universe, tH , is comparable or longer than the relaxation time trelax ≤ tH , it is equivalent to the following condition R≤ tH η ln 0.1 M η 2/3 G M 1/3 , (1.5.4) where N = M/η. (1.5.5) Figure 1.5.1 shows the nuclear stellar clusters as red circles, the well-resolved MBHs as black circles, and the unresolved MBHs as white circles. The black star is M87’s black hole shadow. The black line denotes the positions of the Schwarzschild radius as a function of mass. The solid blue line represents the condition given by equation 1.5.2 for tH = 1010 years (ρ̂crit ≈ 107 M pc−7/3 for solar mass stars, η = 1). The horizontal green line represents the condition given by equation 1.5.3 (≈ 3.5 × 108 M ). The dashed green line denotes the condition given by equation 1.5.4 for tH = 1010 years, which intersects with the solid blue line at the same critical mass determined by equation 1.5.3. The positions of NSCs are restricted within the boundaries defined by the collisional stable region for NSCs, denoted by the thick blue and green lines. It also explains that NSCs will be unstable for masses larger than 108 M which would explain the absence of NSCs with these masses. 1.5. Comparison with theoretical expectations for nuclear clusters at galaxy 14 centers Figure 1.5.1: Measured masses versus effective radius diagram. From: (Escala, 2021). NSC −1 Figure 1.5.2 shows the observed black hole formation efficiency BH = (1+ M ) MBH or what is the same BH = MBH /MCMO as a function of the total mass in the central massive object MCMO = MNSC + MBH , with both quantities computed using the MBHs and NSCs masses from the dataset by (Neumayer et al., 2020), represented by black circles. The efficiency has two dominant values for black hole efficiencies (BH ≤ 0.15 at MCMO ≤ 3 · 107 M and BH ≥ 0.9 for MCMO ≥ 3 · 108 M ) and a transition close to a step function of the mass. Upper limits for the efficiencies BH are denoted by lower triangles, while lower limits for BH by upper triangles, displaying the same trend of the black circles but with larger scatter at lower CMO masses. 1.5. Comparison with theoretical expectations for nuclear clusters at galaxy centers 15 Figure 1.5.2: Mass of the CMO versus the observed efficiency of the black holes formation diagram. Source: (Escala, 2021). Figure 1.5.3 shows the collision and relaxation timescales in the mass versus radius diagram for clusters composed of solar mass stars (η = 1), for different cluster ages tH : 1010 yr in blue, 108 yr in green and 106 yr in purple. For each color with a different tH , clusters on the left side of the solid curves fulfill the condition tcoll ≤ tH or trelax ≤ tH , with the intersection of solid curves dividing the stable trelax ≤ tcoll and the unstable tcoll ≤ trelax regions. The yellow line denotes the same conditions for 100 M Pop III stars with tH = 106 yr. The dotted black line denotes the condition trelax = tcoll for clusters composed of solar mass stars (η = 1) and the dotted red, for clusters of more massive Pop III stars (η = 100). The collision times of the NSCs measured today can be assumed to be longer than they were at the formation. 1.5. Comparison with theoretical expectations for nuclear clusters at galaxy 16 centers Figure 1.5.3: Collision and relaxation timescales in the mass versus radius diagram. Source: (Escala, 2021). Chapter 2. Methodology 17 Chapter 2 Methodology 2.1 NBODY6++ To perform the simulations of this thesis we used the NBODY6++ GPU code, designed to speed up N-body simulations of large numbers of particles (Wang et al., 2015). The code NBODY6++ GPU directly integrates Newton’s equations of motion for many bodies very precisely, giving us information on the dynamic evolution of star clusters. It is used to simulate systems that collide with high precision and a long integration time. This code has four main characteristics: it is based on a fourth-order prediction–correction method (Hermite scheme), individual and block time–steps, a neighbor scheme (Ahmad–Cohen scheme), a regularization scheme for close encounters (KS-Regularization), and few-body subsystems which are described in the manual for the computer code written by (Khalisi et al., 2019). For further discussion of previous versions of the NBODY code see (Aarseth, 1999, 2001). 2.1.1 Hermite scheme Each particle is determined by its velocity v0 , mass m and position r0 at time t0 . The acceleration equation and its time derivative due to all the other particles are given for a particle i by a0,i = − X i6=j Gmj R , R3 (2.1.1) 18 2.1. NBODY6++ ȧ0,i = − X Gmj i6=j V 3R(V · R) , + R3 R5 (2.1.2) where G is the gravitational constant; R = r0,i − r0,j is the relative coordinate; R=\|r0,i − r0,j \| the modulus; and V = v0,i − v0,j the relative space velocity to the particle j. The Hermite scheme follows the trajectory of the particle by first predicting a new position and velocity for the next time step t using the Taylor series for ri (t) and vi (t) given by rp,i (t) = r0 + v0 (t − t0 ) + a0,i (t − t0 )2 (t − t0 )3 + ȧ0,i , 2 6 (2.1.3) (t − t0 )2 . 2 (2.1.4) vp,i (t) = v0 + a0,i (t − t0 ) + ȧ0,i (But so far the requirements for an accurate high-order integrator are still missing.) Then, an improvement is made using Hermite interpolation which approximates the higher accelerating terms by another Taylor series: 1 (3) 1 (2) ai (t) = a0,i + ȧ0,i · (t − t0 ) + a0,i · (t − t0 )2 + a0,i · (t − t0 )3 , 2 6 (2.1.5) 1 (3) (2) ȧi (t) = ȧ0,i + a0,i · (t − t0 ) + a0,i · (t − t0 )2 . 2 (2) (2.1.6) (3) From equations 2.1.5 and 2.1.6 we need to know the terms a0,i and a0,i . They can be calculated using rp and vp following the calculation of the equations 2.1.1 and 2.1.2 and they will be ap,i and ȧp,i respectively. We replace the values of ap,i and ȧp,i on the left-hand side of the equation 2.1.5 and 2.1.6, after simplifying we obtain (3) a0,i − ap,i ȧ0,i + ȧp,i +6 , 3 (t − t0 ) (t − t0 )2 (2.1.7) a0,i − ap,i 2ȧ0,i + ȧp,i −2 . 2 (t − t0 ) (t − t0 ) (2.1.8) a0,i = 12 (2) a0,i = −6 Finally, to have a ’corrected’ position r1,i and velocity v1,i of the particle i at the computation time t1 we need to extend the Taylor series for ri (t) and vi (t) from equations 2.1.3 and 2.1.4 by two more orders (2) (t − t0 ) r1,i (t) = rp,i (t) + a0,i 24 4 + a30,i (t − t0 )5 , 120 (2.1.9) 2.1. NBODY6++ v1,i (t) = vp,i (t) + a20,i 19 4 (t − t0 )3 (3) (t − t0 ) + a0,i . 6 24 (2.1.10) This way we obtain the Hermite scheme able to obtain high-order accelerations from the combination of low-order terms with only the first derivative of the equations. The local error in r and v within the two-time steps ∆ = t1 − t0 is expected to be of order ϑ(∆t5 ), and the global error for a fixed physical integrator time scales with ϑ(∆t4 ) (Makino, 1991). 2.1.2 Individual and block time steps So far we understand that stellar systems have a huge dynamical range in radial and temporal scales with time scales from periods of some days for binaries to relaxation times of star clusters of billions of years. There typically is a large dynamic range in the average local stellar density from the center to the very outskirts of clusters, where it dissolves into the galactic tidal field. If we consider the timestep required for the two closest bodies they would determine the time-step of the force calculation for the whole rest of the system. Then, if we choose a small time step the evolution of the system will be very time-consuming which would not allow us to evolve the simulation for a long enough time. And if we choose a larger time step close encounters will not be calculated correctly. So the idea here is to mix both the small time steps and the larger time steps for the whole system; therefore for large changes, the force is calculated via small time steps, and for small changes, the force is applied on larger time steps. First, the individual time-step scheme (Aarseth, 1963) was studied, and then the block time-steps. Each particle in NBODY6++ GPU is assigned its own ∆ti like in the block time steps in figure 2.1.1. The particle i has the smallest time step at the beginning of the simulation, so its phase space coordinates are calculated at each time step. The particle k has a time step that is twice the time-step for the particle i, at the odd time steps the full force calculation is just ’predicted’ and a full force calculation is due at the dotted lines. The step width may be altered or not after the end of the integration cycle for a particle for example for k and l beyond the label ’8’. The time steps have to stay commensurable with both each other as well as the total time, such that the hierarchy is guaranteed. This is what is called a block step scheme. 20 2.1. NBODY6++ Figure 2.1.1: Illustration of the block time step scheme in NBODY6++ GPU for four particles. Source: NBODY6++ GPU Manual for the computer code (Khalisi et al., 2019). You can find this document here https://github.com/nbodyx/ Nbody6ppGPU/tree/master/doc. A first estimate for the time step could be the rate of change of the acceleration p ∆ti ∝ ai /ȧi . But it turns out that for special situations in many body systems, it provides some undesired numerical errors. After some experiments, the following formula was adopted (Aarseth, 1985). v u 2 u \|a1,i \|\|a(2) 1,i \| + \|ȧ1,i \| , ∆ti = tη (3) (2) \|ȧ1,i \|\|a1,i \| + \|a1,i \|2 (2.1.11) where η is a dimensionless accuracy parameter that controls the error. In most applications, it is taken to be η ≈ 0.01 to 0.02. It can be noticed that after some time there will be a grouping of particles (block) due to the movement at each time step. 2.1.3 Ahmad–Cohen scheme As we consider more particles in the simulations it will take much longer to calculate the full force for each particle within the system. Therefore, it is desirable to use a method to speed up the calculations while retaining the collisional approach. 2.1. NBODY6++ 21 One way to achieve this is to employ the ’neighbor scheme’ suggested by (Ahmad and Cohen, 1973). The idea of the Ahmad-Cohen scheme is to split the force polynomial from equation 2.1.5 on a given particle i into two parts, an irregular and a regular component: ai = ai,irr + ai,reg . (2.1.12) The irregular acceleration ai,irr due to the particles in the neighborhood of a certain particle i (in the code, FI and FIDOT are the irregular force and its time derivative at the last irregular step; internally some routines use FIRR and FD as a local variable). As these particles are close, they give rise to a stronger fluctuating gravitational force, then it is calculated more frequently than the regular one of the more distant particles that don’t change their relative distance to i so quickly (in the code, FR and FRDOT are the regular force and its time derivative at the last regular step; some routines use as a local variable FREG and FDR). The total calculation in equation 2.1.1 can be replaced by a summation over the nearest particles Nnb for ai,irr and adding a distant contribution from all the others. This contribution is updated using another Taylor series FRDOT (that is the time derivative at the last regular step) and the time derivative of FR (the regular force at the last regular step) at the last regular force computation. If a particle is a neighbor or not is determined by its distance. All particles inside a specified ’neighbor sphere’ with a radius rs as we can see in figure 2.1.2 (Makino and Aarseth, 1992) are kept in a list, which is modified at the end of each regular time step when a total force calculation is carried out. Particles outside this sphere will be calculated for their irregular force FIRR. Also, approaching particles within a surrounding shell satisfying R · V < 0 are included. This ’buffer zone’ serves to identify fast-approaching particles before they come in too far into the neighbor sphere. The neighbor criterion is determined by the relative forces between the particles, not by their distance. The filled dots inside the sphere are in the list that is filled in to calculate the total force at the beginning of the simulation. The irregular components are calculated first and then the regular ones. At the same time also the first-time derivative is calculated. From the equations 2.1.5 and 2.1.6 the position and velocity of the particle i are predicted. 22 2.1. NBODY6++ Figure 2.1.2: A neighbor scheme for a particle marked as the asterisk. Source: NBODY6++ GPU Manual for the computer code (Khalisi et al., 2019). As we can see in figure 2.1.3 at the time t1,irr the "corrector" is applied only for the irregular acceleration from the neighbors; the regular acceleration is not corrected, but obtained by extrapolating. At the time t2,irr ocurrs the same. The Hermite predictor-corrector method is also applied and another new list of neighbors is created. Therefore, at certain times only the forces from neighbors (irregular time steps) are calculated, while at other times both the forces from neighbors and distant particles (regular time steps) are calculated. In the code NBODY6++ GPU the variable NNBOPT controls the size of the neighbor spheres. Its typical values are between 50 and 200 for a very wide range of total particle numbers. From equation 2.1.11 the accuracy can be tuned by ηirr ≈ 0.01 and ηreg ≈ 0.02 again. 2.1. NBODY6++ 23 Figure 2.1.3: Illustration of the regular and irregular time steps. Source: NBODY6++ GPU Manual for the computer code (Khalisi et al., 2019). 2.1.4 Regularization of close encounters and few-body subsystems The small time steps and small distances in the dynamics of close binaries are treated via regularization. This is one of the most expensive calculations within NBODY6++ GPU. The impact parameter determines the close encounters and is smaller than the parameter for a 90-degree deflection, 2 p90 = 2G(m1 + m2 )/v∞ , (2.1.13) where G, m1 , m2 , and v∞ are the gravitational constant, the masses of the two particles, and their relative velocity at infinity. These close encounters occur more in the center of the cluster since in this place the distances between particles are smaller. What happens is that the relative distances between the particles are very small and their time steps are also very small, which causes the error to grow in these places due to the singularity in the gravitational potentials. This is the problem that regularization resolves. The regularization defines two parameters within NBODY6++ GPU: RMIN and DTMIN. RMIN indicates the maximum separation for a close encounter and when these particles are closer than RMIN (from equation 2.1.13) and the time steps are smaller than DTMIN then the 24 2.1. NBODY6++ conditions for regularization are fulfilled. DTMIN is defined as: h η i r3 1/2 min , dtmin = κ 0.03 hmi (2.1.14) where κ is a free numerical factor, η the general time step factor, and <m> the average stellar mass. That is why regularization is an elegant trick to deal with particles that are as close as the diamond in figure 2.1.2. The principal idea is to take both stars out of the main integration cycle, replace them by their center of mass and advance the usual integration with a composite particle instead of resolving the two components. The two members of the regularized pair (henceforth KS pair) will be relocated to the beginning of all vectors containing particle data, while at the end one additional center of mass (c.m.) particle is created. One of the purposes of the code variable NAME(I) is to identify particles after such a reshuffling of data. Also, the two particles have to fulfill two more sufficient criteria: that they are approaching each other, and that their mutual force is dominant. In the equations in routine search.f, these sufficient criteria are defined as: p R · V > 0.1 (G(m1 + m2 )R), γ := \|apert \| · R2 < 0.25, G(m1 + m2 ) (2.1.15) (2.1.16) where apert is the vectorial differential force exerted by other perturbing particles onto the two candidates, and R, R, V are scalar and vectorial distance and relative velocity vector between the two candidates, respectively. The factor 0.1 in the upper equation allows nearly circular orbits to be regularized. The condition γ < 0.25 demands that the relative strength of the perturbing forces compared to the pairwise force is one-quarter at most. Then these subsystems are separated from the rest of the system but not unperturbed. After this, the motion of the new particle is put into a new coordinate system (three-dimensional cartesian space). Any unperturbed two–body orbit in real space is mapped onto a harmonic oscillator in KS–space with double the frequency. The internal time-step of such a KS-regularized pair depends on the parameter ETAU, which is of the order of some 50-100 steps per orbit. The first studies on this topic were done in (Stiefel, 1965). A more modern theoretical version can be found at (Neutsch and Scherer, 1992). The Hamiltonian formalism is explained in 2.1. NBODY6++ 25 (Mikkola, 1997). When the perturbation γ falls below a critical value of the input parameter GMIN ≈ 10−6 , it is considered to be unperturbed and the analytical solution for the Keplerian orbit is used instead of doing numerical integration. A little bit misleading is that such unperturbed KS–pairs are denoted in the code as ”mergers”, e.g. in the number or merges (NM) and the energy of the mergers (EMERGE). Merged pairs can be resolved at any time if the perturbation changes. The two–body KS regularization occurs in the code either for short-lived hyperbolic encounters or for persistent binaries. In the code, the KS–pair appears as a new particle at the position of the center of mass. The variable NTOT which contains the total number of particles N plus the c.m.’s is increased by 1. When the pair is disrupted, NTOT has decreased again. The maximum number of possible KS– pairs are saved in the variable KMAX, which sets a threshold for the extension of the vector NTOT. Close encounters between single particles and binary stars are also a central feature of cluster dynamics. Such temporary triple systems often reveal irregular motions, ranging from just a perturbed encounter to a very complex interaction, in which disruption of binaries, exchange of components, and ejection of one star may occur. Although not analytically solvable, the general three–body problem has received much attention. So, the KS–regularization was expanded to the isolated 3– and 4–body problem, and later on to the perturbed 3–, 4–, and finally to the N–body problem. The routines are called triple.f, quad.f, chain.f. While occurrences of “triple” and “quad” will be rare in a simulation, the chain regularization is invoked if a KS–pair has a close encounter with another single star or another pair. Especially if systems start with a large number of primordial binaries, such encounters may lead to stable (or quasi-stable) hierarchical triples, quadruples, and higher multiples. They have to be treated by using special stability criteria. A typical way to treat all such special higher subsystems is to define their c.m. to be a pseudo-particle, i.e. a particle with a known sub-structure. The members of the pseudo-particles will be deactivated by setting their mass to zero (ghost particles). At present, there can only be one chain at a time in the code, while merged KS binaries and hierarchical subsystems can be more frequent. Every subsystem — KS pair, chain or hierarchical subsystem is perturbed. Perturbers are typically those objects that get closer to the object than Rsep 26 2.1. NBODY6++ 1/3 = R/γmin , where R is the typical size of the subsystem; for perturbers, the components of the subsystem are resolved in their force computation as well (routines cmfreg.f, cmfirr.f). Figure 2.1.4: Flow chart NBODY6++ GPU. Source: NBODY6++ GPU Manual for the computer code (Khalisi et al., 2019). 2.1. NBODY6++ 27 Figure 2.1.5: Flow chart NBODY6++ GPU. Source: NBODY6++ GPU Manual for the computer code (Khalisi et al., 2019). In the figures 2.1.4 and 2.1.5 we can see the general structure of how the NBODY6++ GPU code works depending on the parameters defined in the input file. 28 2.2. Description of the numerical setup 2.2 Description of the numerical setup 2.2.1 Input We model dense star clusters in virial equilibrium using a Plummer distribution for the stars (Plummer, 1911). Half of our simulations included an analytic background potential, which helps us simulate the effects of the gas within the cluster, which also follows a Plummer density profile. This is a template of the input file used to carry out the simulations with NBODY6++ GPU. The file was used for all simulations with only minor changes. These small changes are the termination time in N-body units (TCRIT), the virial radius of the cluster (RBAR), the mean mass of the stars (ZMBAR), and the maximum and minimum particle mass (BODY1 and BODYN). The last three parameters have to be the same because we are not considering stellar evolution nor an initial mass function therefore the masses are the same throughout the evolution of the cluster, and the initial radius of the stars in the file instar.F which is in the folder ’.../src/Main/’. The NBODY code uses dimensionless units with the name Nbody units. More detail on how these different variables are defined within the code can be found in (Khalisi et al., 2019). The input file contains more than 90 parameters that must be adjusted for each simulation. The names of the different input parameters are described below: Line 1: KSTART, TCOMP, TCRITp, isernb, iserreg, iserks. Line 2: N, NFIX, NCRIT, NRAND, NNBOPT, NRUN. Line 3: ETAI, ETAR, RSO, DTADJ, DELTAT, TCRIT, QE, RBAR, ZMBAR. Line 4: KZ(1), KZ(2), KZ(3), KZ(4), KZ(5), KZ(6), KZ(7), KZ(8), KZ(9), KZ(10). Line 5: KZ(11), KZ(12), KZ(13), KZ(14), KZ(15), KZ(16), KZ(17), KZ(18), KZ(19), KZ(20). Line 6: KZ(21), KZ(22), KZ(23), KZ(24), KZ(25), KZ(26), KZ(27), KZ(28), KZ(29), KZ(30). Line 7: KZ(31), KZ(32), KZ(33), KZ(34), KZ(35), KZ(36), KZ(37), KZ(38), KZ(39), KZ(40). Line 8: KZ(41), KZ(42), KZ(43), KZ(44), KZ(45), KZ(46), KZ(47), KZ(48), 2.2. Description of the numerical setup 29 KZ(49), KZ(50). Line 9: DTMIN, RMIN, ETAU, ECLOSE, GMIN, GMAX, SMAX. Line 10: AlPHA, BODY1, BODYN, NBINO, NHIO, ZMET, EPOCHO, DTPLOT. Line 11: Q, VXROT, VZROT, RTIDE. Line 12: MP, AP, MPDOT, TDELAY. Line 1 contains the parameters that are defined in the file nbody6.F. The parameters of lines 2, 3, 4, 5, 6, 7, 8, and 9 are defined in the file input.F. The parameters of line 10 are defined in the file data.F. Scale.F contains the definitions of the parameters of line 11 and finally xtrn10.F includes the definition of the parameters of line 12. The input file for NBODY6++ GPU is written as follows. For the case with an external potential: 1 1000000.0 1.E6 40 40 0 10000 1 10 43532 190 1 10 0.02 0.01 0.25 1.0 1.0 48000.0 2.0E00 0.05 15.0 0110104012 0104000430 1 1 2 0 0 2 -1 0 0 2 1020001100 0 0 0 0 0 4 -2 0 1 0 1.0E-03 1E-3 0.2 1.0 1.0E-06 0.01 1 1.0 15.0 15.0 0 0 0.0001 0 1.0 0.5 0.0 0.0 0.0 1.0 0.59 10000 10000 For the case without an external potential: 1 1000000.0 1.E6 40 40 0 10000 1 10 43532 100 1 10 0.02 0.01 0.17 1.0 1.0 24000.0 2.0E00 0.05 15.0 30 2.2. Description of the numerical setup 0110104012 0100000430 1 1 2 0 0 2 -1 0 0 2 1020001100 0 0 0 0 0 4 -2 0 1 0 1.0E-03 1E-3 0.2 1.0 1.0E-06 0.01 1 1.0 15.0 15.0 0 0 0.0001 0 1.0 0.5 0.0 0.0 0.0 We use a version of the code modified by Bastian Reinoso to include stellar collisions avoiding the use of the stellar evolution routines from the original version. Therefore the mass of the stars is always the same during the evolution of the cluster unless they collide. The simulated cluster has a Plummer distribution. Some simulations include an external analytic Plummer potential. The external analytic Plummer potential can be added on line 5 in column number 4 which corresponds to KZ(14)=4 with an additional line at the end of the input, which is MP that is the total mass of the Plummer sphere in scaled units, AP the Plummer scale factor in N-body units, MPDOT the decay time for gas expulsion and TDELAY the delay time for starting gas expulsion. The evolution of clusters was followed until 10 Myr. The example used is a simulation of 10,000 particles with a Plummer potential until N-body units. Since we don’t consider stellar evolution, the stellar mass at the beginning of the simulation and the final stellar mass are the same, i.e. ZMBAR=BODY1=BODYN, which in this case is equal to 15 M . The virial radius is 0.05 [pc] and the stellar radius is 1.51 R while the radius of the external potential Rv,ext is the same as the virial radius of the stellar distribution. The crossing time is the time required for a star with a typical velocity to cross the system. The crossing time for a system in virial equilibrium without an external potential is defined by s tcross = Rv3 , GMstars (2.2.1) where Rv is the virial radius of the cluster, G is the gravitationsl constant and M is the total mass of the stars. The relaxation time is the time needed for a system to return to a state of 2.2. Description of the numerical setup 31 equilibrium. The half-mass relaxation time for a system with identical particles is defined as trh = 0.138 N tcross ln(γN ) (2.2.2) in the absence of an external potential. The crossing time including an external potential is defined by s tcross,ext = Rv3 1 . GMstars 1 + q (2.2.3) The core collapse time scale is related to the crossing time as tcc ∝ (1 + q)4 tcross . ext . Here q is defined as q = MMstars Then, the half-mass relaxation time with an external potential can be calculated as trh,ext = 0.138 N (1 + q)4 tcross,ext , ln(γN ) (2.2.4) where γ = 0.4 for equal-mass stars. 2.2.2 Initial conditions The table 2.2.1 details the initial conditions of each simulation made in this thesis in which all simulations have 104 stars. The first column is the number of the simulation, the second column is the initial mass of the stars in solar masses, the third column is the initial radius of the stars in solar radius, the fourth column is the virial radius of the cluster in parsecs, the fifth column is the total mass of the cluster in solar masses, the sixth column is the mass of the gas in solar masses, the seventh column is the radius of the gas in pc and the eighth column is the crossing time in Myr. It is important to mention that the initial radii and masses are from the paper of (Windhorst et al., 2018) and the stars in the clusters have the same mass and radius initially. 32 2.3. Collision treatment ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Mstar [M ] 15 30 50 100 15 30 50 100 15 30 50 100 15 30 50 100 15 30 50 100 15 30 50 100 Rstar [R ] 1.51 2.12 2.86 4.12 1.51 2.12 2.86 4.12 1.51 2.12 2.86 4.12 1.51 2.12 2.86 4.12 1.51 2.12 2.86 4.12 1.51 2.12 2.86 4.12 Rv [pc] 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Mcluster [M ] 3 × 105 6 × 105 1 × 106 2 × 106 1.5 × 105 3 × 105 5 × 105 1 × 106 3 × 105 6 × 105 1 × 106 2 × 106 1.5 × 105 3 × 105 5 × 105 1 × 106 3 × 105 6 × 105 1 × 106 2 × 106 1.5 × 105 3 × 105 5 × 105 1 × 106 Mgas [M ] 1.5 × 105 3 × 105 5 × 105 1 × 106 — — — — 1.5 × 105 3 × 105 5 × 105 1 × 106 — — — — 1.5 × 105 3 × 105 5 × 105 1 × 106 — —— — Rgas [pc] 0.05 0.05 0.05 0.05 — — — — 0.1 0.1 0.1 0.1 — — — — 0.5 0.5 0.5 0.5 — — — — tcross [Myr] 2.1 × 10−4 1.5 × 10−4 1.1 × 10−4 8.0 × 10−5 4.3 × 10−4 3.0 × 10−4 2.3 × 10−4 1.6 × 10−4 6.0 × 10−4 4.3 × 10−4 3.3 × 10−4 2.3 × 10−4 1.2 × 10−3 8.6 × 10−4 6.6 × 10−4 4.7 × 10−4 6.5 × 10−3 4.8 × 10−3 3.7 × 10−3 2.6 × 10−3 1.3 × 10−2 9.6 × 10−3 7.4 × 10−3 5.2 × 10−3 Table 2.2.1: Initial conditions of the simulations. 2.3 Collision treatment The stellar collision condition used is: r ≤ R1 + R2 , (2.3.1) where r is the distance between both stars center of mass and R1 and R2 are the radii of the stars. A collision occurs when two stars are separated by a distance equal to or smaller than the sum of their radii, which also means that they are in physical contact. When this condition is reached, then both stars are replaced by a new star whose total mass is the sum of the masses of the stars before the collision. Thus, the new star assumes the same density as the previously merging 2.3. Collision treatment 33 stars. The mass and radius of the new star are given by the following equations Mnew = M1 + M2 , Rnew = R1 M1 + M2 M1 (2.3.2) 1/3 , (2.3.3) where M1 and M2 are the mass of the two stars. The initial mass of the cluster Mcluster is related to the number of stars N and the mass of the star Mstar Mstar = Mcluster /N (2.3.4) The total mass of the cluster with an external potential is calculated as Mtot = Mstars + Mext . (2.3.5) 34 Chapter 3. Analysis Chapter 3 Analysis 3.1 Pop. III star cluster simulations In this work, we run 24 simulations, and the evolution of all the clusters in our sample evolved up to 10 Myr to be able to be analyzed later in the mass versus radius diagrams. All clusters simulated here have a total of 104 stars and in each cluster, the mass of the external potentials is equal to the mass in stars (Mext = Mstars ). Analyzing the clusters to which we included a background potential, we noticed that they have in common that regardless of their initial conditions, only a single star escapes at the end of their evolution of these. It is important to note that the words ’very massive stars’ are included in the following descriptions because the evolution of these clusters shows single collisions (a single collision at different times with different objects within the cluster). For the simulations 17, 18, 19, and 20 (ID 17, ID 18, ID 19, and ID 20) do not dominate collisions (tcoll > trelax ) which does not result in the formation of a most massive object but rather the formation of several very massive stars (since the single collisions are not with the same object). These clusters have in common that they have an external potential and that they have a virial radius of Rv = 0.5 pc. ID17 has a mass in the cluster of Mcluster = 3 × 105 M , with each star having a mass equal to Mstar = 15 M , a stellar radius of Rstar = 1.51 R and an external potential of Mgas = 1.5 × 105 M . ID 18 has a mass of the cluster equal to Mcluster = 6 × 105 M , with each star having a mass equal to Mstar = 30 M , a stellar radius of Rstar = 2.12 R and an external potential of Mgas = 3 × 105 M . For ID 19 the total mass of the cluster is equal to Mcluster = 1 × 106 M , 3.1. Pop. III star cluster simulations 35 with each star having a mass equal to Mstar = 50 M , a radius of Rstar = 2.86 R and an external potential of Mgas = 5 × 105 M . ID 20 has a mass of this cluster equal to Mcluster = 2 × 106 M , with each star having a mass equal to Mstar = 100 M , a radius of Rstar = 4.12 R and an external potential of Mgas = 1 × 106 M . All these simulations have in common that within a cluster that is mostly stable with horizontal Lagrangian radii lines with a slight expansion of the cluster for the different percentages of enclosed mass. The following set of simulations 9, 10, 11, and 12 (ID 9, ID 10, ID 11, and ID 12) have a virial radius equal to Rv = 0.1 pc and also an external potential. ID 9 has stars of equal mass of Mstar = 15 M , a radius of Rstar = 1.51 R , a total mass of the cluster of Mcluster = 3 × 105 M and an external potential of Mgas = 1.5 × 105 M . ID 10 has stars of equal mass of Mstar = 30 M , radius of Rstar = 2.12 R , a total mass of the cluster of Mcluster = 6 × 105 M and an external potential of Mgas = 3 × 105 M . ID 11 has stars of equal mass of Mstar = 50 M , a radius of Rstar = 2.86 R , a total mass of the cluster of Mcluster = 1 × 106 M and an external potential of Mgas = 5 × 105 M . The cluster in ID 12 has stars of an equal mass of Mstar = 100 M , a radius of Rstar = 4.12 R , a total mass of the cluster of Mcluster = 2 × 106 M and an external potential of Mgas = 1 × 106 M . The mass of the most massive object for ID 9 achieves a mass of 2775 M after the collapse at ∼ 6 Myr, for ID 10 achieves a mass of 13920 M after the collapse ∼ 4 Myr, in ID 11 it reaches a mass of 33700 M where there is a collapse between 2.5 and 3.5 Myr and the cluster in ID 12 reaches a mass of 96100 M with a collapse at ∼ 2 Myr. In all this set of simulations, there is an expansion of the lagrangian radii for 10%, 50%, and 90% of the enclosed mass after only one collapse during its evolution. In figure 3.1.1, we show simulation number 2 (ID2) with an external potential where the collapse of the cluster occurs between 1 and 2 Myr. In those times, the largest number of collisions in the cluster occurs, which makes the mass of the massive object grow up to 26190 M . In the simulations with potential, a clear delay in the collapse of the cluster is seen in the work previously done by Reinoso et al. (2020) which makes the crossing time vary by 0.5 in the specific case that the mass of the external potential and the total mass in stars are equal inside the cluster compared to simulations without this external potential. This delay is mainly due to the increase in the kinetic energy of the stars within the cluster, which makes the gravitational collapse take a little longer to occur. This is more 36 3.1. Pop. III star cluster simulations visible when analyzing the simulations with and without the potential for the same physical parameters. The Lagrangian radii show us that after the collapse there is an expansion of the cluster at 10%, 50%, and 90% of the enclosed mass. Figure 3.1.1: Evolution of a star cluster with 104 stars with an external potential of the same mass. Each star in the cluster has the same mass and radius of 30 M and 2.12 R , respectively. The virial radius is 0.05 pc and the cluster has evolved to 10 Myr. The first panel shows the number of collisions over time, the second panel shows the evolution of the Lagrangian radii of the cluster at 10%, 50% and 90% of the enclosed mass, the third panel shows the mass of the most massive object in the cluster divided by the initial mass of each star and the last panel shows the number of escaping stars divided by the total number of stars. Source: Kirsty L.K. Sehlke-Abarca et. al. in preparation. With the same virial radius Rv = 0.05 pc and with a background potential are the simulations number 1 (ID 1), number 3 (ID 3), and number 4 (ID 4). ID 1 has stars of equal mass of Mstar = 15 M , a radius of Rstar = 1.51 R , a total mass of the cluster of Mcluster = 3 × 105 M and an external potential of Mgas = 1.5 × 105 M . ID 3 has stars of equal mass of Mstar = 50 M , a radius of Rstar = 2.86 R , a total mass of the cluster of Mcluster = 1 × 106 M and an external potential of 3.1. Pop. III star cluster simulations 37 Mgas = 5 × 105 M . The densest simulation of this work is ID 4 which has stars of an equal mass of Mstar = 100 M , a radius of Rstar = 4.12 R , a total mass of the cluster of Mcluster = 2 × 106 M and has an external potential of Mgas = 1 × 106 M . These clusters reach a mass of the most massive object of 11385 M collapsing at ∼ 2 Myr for ID 1, 59600 M for ID 3 collapsing at ∼ 1 Myr and a mass of 171400 M for the densest cluster ID 4 that a collapse takes place between 0 and 1 Myr. The ID 1 cluster shows an expansion of the Lagrangian radii for 10%, 50%, and 90% of the enclosed mass while for ID 3 and ID 4, there is a collapse of the innermost layer shown via the 10% line and an expansion of the outermost layers such as the 50% and 90% line of the enclosed mass. For the simulations 21, 22, 23, and 24 (ID 21, ID 22, ID 23, and ID 24) do not dominate collisions (tcoll > trelax ). Clusters in this sample that do not have a background potential and have a viral radius of Rv = 0.5 pc are described below. The only simulations in which there was no very massive object were simulations number 21 and 22 (ID 21 and ID 22) since there are no collisions in these clusters. In these cases, the cluster expands slightly with some escaping stars being 13 and 27 respectively. ID 21 has a total mass of Mcluster = 1.5 × 105 M , stars of equal mass of Mstar = 15 M and stellar radii of Rstar = 1.51 R and ID 22 has a total mass of Mcluster = 3 × 105 M , stars of equal mass of Mstar = 30 M and stellar radii of Rstar = 2.12 R . For simulation number 23 (ID 23) there are few collisions and very massive stars are formed. This cluster is expanding slightly and the stars escaping is about 55. It should be noted that this cluster is characterized by having a total mass of Mcluster = 5 × 105 M , stars of an equal mass of Mstar = 50 M and stellar radii of Rstar = 2.86 R . Finally, in simulation number 24 (ID 24) there are few collisions and very massive stars are formed. This cluster is also expanding slightly and the stars escaping is about 132. It should be noted that this cluster is characterized by having a total mass of Mcluster = 1 × 106 M , stars of an equal mass of Mstar = 100 M and radius of Rstar = 4.12 R . The following set of simulations 13, 14, 15, and 16 (ID 13, ID 14, ID 15, and ID 16) has a viral radius of Rv = 0.1 pc and do not have a background potential. The collapse of ID 13 occurs at ∼ 2.5 Myr, in which the total mass of the cluster is Mcluster = 1.5 × 105 M , stars of an equal mass of Mstar = 15 M and radius of Rstar = 1.51 R . The mass of the most massive object reaches 5895 M and stars equal to 826 escape from the cluster, therefore a mass of 131715 M remains in the cluster. The collapse of ID 14 occurs at ∼ 1.5 Myr, in which the total 38 3.1. Pop. III star cluster simulations mass of the cluster is Mcluster = 3 × 105 M , stars of an equal mass of Mstar = 30 M and radius of Rstar = 2.12 R . The mass of the most massive object reaches 16200 M and stars equal to 1078 escapes from the cluster, therefore a mass of 251460 M remains in the cluster. The collapse of ID 15 occurs at ∼ 1 Myr, in which the total mass of the cluster is Mcluster = 5 × 105 M , stars of an equal mass of Mstar = 50 M and radius of Rstar = 2.86 R . The mass of the most massive object reaches 33650 M and stars equal to 1326 escapes from the cluster, therefore a mass of 400050 M remains in the cluster. The collapse of ID 16 occurs before 1 Myr. Here the total mass of the cluster is Mcluster = 1 × 106 M , stars of equal mass of Mstar = 100 M and stellar radii of Rstar = 4.12 R . The mass of the most massive object reaches 89900 M and stars equal to 1604 escapes from the cluster, therefore a mass of 749700 M remains in the cluster. In all these simulations there is also an expansion of the outer layers of the Lagrangian radii that encompass the enclosed mass at 50% and 90% of the enclosed mass. In figure 3.1.2, we show simulation number 6 (ID6). As mentioned above for these simulations the collapse occurs earlier, in this case before 1 Myr. The number of collisions in this simulation is higher than in the simulation shown in the previous figure. This makes the growth of the central object much faster until 28800 M . A collapse of the shell at 10% of the Lagrangian radius and an expansion of the shells for 50% and 90% are shown. The amount of stars that escape in simulations without potential is much higher than those that escape in simulations with potential with 1981 stars. 3.1. Pop. III star cluster simulations 39 Figure 3.1.2: Evolution of a star cluster of 104 stars without an external potential, each star in the cluster has the same mass and radius 30 M and 2.12 R , respectively. The virial radius is 0.05 pc and the cluster has evolved to 10 Myr. The first panel shows the number of collisions over time, the second panel shows the evolution of the Lagrangian radii of the cluster at 10%, 50% and 90% of the mass, the third panel shows the mass of the most massive object in the cluster divided by the initial mass of each star and the last panel shows the number of escaping stars divided by the total number of stars. Source: Kirsty L.K. Sehlke-Abarca et. al. in preparation. With the same virial radius Rv = 0.05 pc and without a background potential are the simulations number 5 (ID 5), number 7 (ID 7), and number 8 (ID 8). ID 5 has stars of an equal mass of Mstar = 15 M , a radius of Rstar = 1.51 R and a total mass of the cluster of Mcluster = 1.5 × 105 M reaching a mass of the most massive object of 12195 M and stars equal to 1633 escape from the cluster, therefore a mass of 113310 M remains in the cluster. The collapse of the cluster occurs before 1 Myr and after that, there is an expansion of the lagrangian radii lines for 50% and 90% of the enclosed mass. ID 7 which has stars of an equal mass of Mstar = 50 M , a stellar radius of Rstar = 2.86 R and a total mass of the cluster of Mcluster = 5 × 105 M reaching a mass of the most massive object of 55900 M 40 3.2. Theoretical results for Pop. III star clusters and stars equal to 2304 escape from the cluster, therefore a mass of 328900 M remains in the cluster. The collapse of the cluster occurs before 1 Myr and there is a first collapse of the innermost layer and then a second collapse deeper also for 10% of the Lagrangian radius and expansion in the outermost layers for 50% and 90% of the Lagrangian radii of the enclosed mass. On the other hand, ID 8 which contains stars of an equal mass of Mstar = 100 M , a radius of Rstar = 4.12 R and a total mass of the cluster of Mcluster = 1 × 106 M reaching a mass of the most massive object of 138200 M and stars equal to 2672 escape from the cluster, therefore a mass of 594600 M remains in the cluster. The collapse of the cluster occurs before 1 Myr and there is a first collapse of the innermost layer and then a second collapse deeper also for 10% of the Lagrangian radius and expansion in the outermost layers for 50% and 90% of the Lagrangian radii of the enclosed mass. 3.2 Theoretical results for Pop. III star clusters In figure 3.2.1 the theoretical results of the simulations for 1Myr can be observed. To make the following three figures, other parameters like the effective crosssection, the Safronov number, and velocity dispersion which are defined later were considered in the initial (Escala, 2021) equations, which causes a change in the figures. For a better understanding, I will rewrite the initial equations with the new parameters in consideration for equation 1.5.2. Star clusters that have tcoll ≤ tH are unable to expand before the onset of runaway collisions, they will probably collapse towards the formation of an MMO: M≥ 4πMstar 3Σ0 tH G1/2 2/3 R7/3 , (3.2.1) where Mstar is the mass of a single star, Σ0 is the effective cross section, tH is the age of the system and G is the gravitational constant. The effective cross section Σ0 √ 2 is defined as Σ0 = 16 πRstar (1 + Θ), where Rstar is the radius of a single star and Θ is the Safronov number Θ = 9.54((Mstar R )/( M Rstar ))((100 km s−1 )/σ))2 . p σ is the velocity dispersion of a virialized system σ = GM/R, where M is the total mass and R the characteristic radius of the system. On the other hand, how Pop. III star clusters (PSCs) that have trelax ≤ tH will expand their effective radius before collisions become important, moving its 3.2. Theoretical results for Pop. III star clusters 41 position to the collisional stable region: R ≤ [((tH Mstar )/0.1) ln (M/Mstar )]2/3 (G/M )1/3 . (3.2.2) From the figures 3.2.1, 3.2.2 and 3.2.3 we can see that if we consider longer times the conditions for the solid lines and the segmented ones move from left to right which means that clusters with a smaller virial radius are more unstable, forming most massive objects more quickly. Figure 3.2.1: In this figure the radius of the cluster in pc is shown on the x-axis while on the y-axis is the mass of the cluster in M . The different colors in the figure represent the different initial stellar masses for both triangles and lines. In this case, yellow represents the initial mass of each star of 15 M , cyan represents the initial mass of each star of 30 M , magenta represents an initial mass of each star of 50 M and finally coral represents an initial mass of each star of 100 M . Triangles pointing to the right (B) represent simulations with an external potential on the other hand triangles pointing to the left (C) represent simulations without an external potential. The solid lines are from the condition of equation 3.2.1 for the different σ associated with the initial conditions, while the dashed lines are from equation 3.2.2 both for a time evolution of 1 Myr. Source: Kirsty L.K. Sehlke-Abarca et. al. in preparation. 42 3.2. Theoretical results for Pop. III star clusters Figure 3.2.2: In this figure the radius of the cluster in pc is shown on the x-axis while on the y-axis is shown the mass of the cluster in M . The different colors in the figure represent the different initial stellar masses for both triangles and lines, in this case, yellow represents an initial mass of each star of 15 M , cyan represents an initial mass of each star of 30 M , magenta represents an initial mass of each star of 50 M and finally coral represents an initial mass of each star of 100 M . Triangles pointing to the right (B) represent simulations with an external potential on the other hand triangles pointing to the left (C) represent simulations without an external potential. The solid lines are from the condition of equation 3.2.1 for the different σ associated with the initial conditions, while the dashed lines are from equation 3.2.2 both for a time evolution of 5 Myr. Source: Kirsty L.K. Sehlke-Abarca et. al. in preparation. 3.3. Efficiencies for Pop. III star clusters 43 Figure 3.2.3: In this figure the radius of the cluster in pc is shown on the x-axis while on the y-axis is shown the mass of the cluster in M . The different colors in the figure represent the different initial stellar masses for both triangles and lines, in this case, yellow represents an initial mass of each star of 15 M , cyan represents an initial mass of each star of 30 M , magenta represents an initial mass of each star of 50 M and finally coral represents an initial mass of each star of 100 M . Triangles pointing to the right (B) represent simulations with an external potential on the other hand triangles pointing to the left (C) represent simulations without an external potential. The solid lines are from the condition of equation 3.2.1 for the different σ associated with the initial conditions, while the dashed lines are from equation 3.2.2 both for a time evolution of 10 Myr. Source: Kirsty L.K. Sehlke-Abarca et. al. in preparation. 3.3 Efficiencies for Pop. III star clusters In figures 3.3.1, 3.3.2 and 3.3.3 showing the efficiencies for the formation of most massive objects we have that the x-axis shows the initial stellar mass of the cluster Mini divided by the critical mass Mcrit and in the y-axis the efficiency 44 3.3. Efficiencies for Pop. III star clusters MMO of each simulation. We have Mini = MPSC,final + MMMO + Mesc , (3.3.1) Figure 3.3.1: Efficiencies at which MMOs are formed calculated with equation 3.3.3. The x-axis shows the initial stellar mass of the cluster Mini divided by the critical mass Mcrit until 1 Myr. Source: Kirsty L.K. Sehlke-Abarca et. al. in preparation. where MPSC,final is the mass of the Pop. III star cluster remaining at the end of the simulation, MMMO is the mass of the most massive object and Mesc is the mass of the stars escaping from the cluster. Here we consider that the mass of the central massive object is equal to MCMO = MPSC + MMMO . On the other hand, the critical mass Mcrit is obtained from the condition given by the equation 3.2.1: Mcrit = 4πMstar 3Σ0 tH G1/2 2/3 R7/3 . (3.3.2) 3.3. Efficiencies for Pop. III star clusters 45 The efficiency MMO is obtained from the same equation used in (Escala, 2021), −1 MPSC MMO = 1 + , MMMO (3.3.3) where the mass of the PSC is calculated as MPSC = (1 − MMO )MCMO and the mass of the MMO can be calculated as MMMO = MMO MCMO . From here we notice that the simulations in which we do not add a background potential have a higher efficiency than when we consider this background potential for the same physical conditions of the cluster. The highest efficiencies are reached for denser and more massive clusters which reach a value of ∼ 20%. Figure 3.3.2: Efficiencies at which MMOs are formed calculated with equation 3.3.3. The x-axis shows the initial stellar mass of the cluster Mini divided by the critical mass Mcrit until 5 Myr. Source: Kirsty L.K. Sehlke-Abarca et. al. in preparation. It is important to note that the mass of the background potential was only considered in the simulations and not in the subsequent calculations of the efficiencies since this potential only affects the dynamics of the cluster and does 46 3.3. Efficiencies for Pop. III star clusters not directly contribute to the growth of the most massive object to be considered in efficiency. The error bars are associated with the average of three simulations for the same single configuration with a different random seed to obtain the statistics of the simulations. Figure 3.3.3: Efficiencies at which MMOs are formed calculated with equation 3.3.3. The x-axis shows the initial stellar mass of the cluster Mini divided by the critical mass Mcrit until 10 Myr. Source: Kirsty L.K. Sehlke-Abarca et. al. in preparation. The highest efficiencies at 10 Myr are obtained from simulations ID4 and ID8 which include the densest and most massive clusters, which reach efficiencies 20 %, since these simulations are located close, in the radius versus mass diagram, to the line that describes the condition (tcoll ≤ tH ) where collisions dominate. The ID7 simulation reaches 16.6 %. A 12.5 % in efficiency is obtained from the ID3 and ID6 simulations. The ID5 simulation reaches a 10 % in efficiency. A little further down the ID2 simulation has 9.09 % in efficiency. Finally, for simulations with a virial radius Rv of 0.05 pc, the ID1 simulation achieves 7.69 % in efficiency. 3.3. Efficiencies for Pop. III star clusters 47 For simulations with a virial radius Rv of 0.1 pc, the ID16 simulation reaches 11.1 % in efficiency. A 10 % in efficiency is obtained by the ID12 simulation. The ID15 simulation has an 8.33 % in efficiency. 7.14 % is obtained from the initial conditions of the ID11 simulation. The ID14 simulation reaches 6.25 %. The ID10 and ID13 simulations have efficiencies of 4.76 % and 4.34 % respectively. And finally, 1.85 % is obtained by the ID9 simulation. The efficiencies of the ID17, ID18, ID19, ID20, ID21, ID22, ID23, ID24 simulations are very close to 0 % for virial radius Rv of 0.5 pc. These last simulations are located close, in the radius versus mass diagram, to the line that describes the condition (trelax ≤ tH ) where collisions do not dominate. 48 3.4 3.4. Summary of the main results Summary of the main results In the following tables 3.4.1,3.4.2 and 3.4.3 we can summarize the main results of the simulations for 1 Myr, 5 Myr, and 10 Myr. ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 MMMO [M ] 30 60 33100 150300 4155 12660 31450 88400 30 60 100 200 30 60 100 22000 30 60 100 200 0 0 50 100 Nesc 1 1 1 1 209 364 492 712 1 1 1 1 10 27 65 186 0 0 0 0 0 0 1 1 MPSC [M ] 149955 299910 466850 849600 142710 276420 443950 840400 149955 299910 499850 999700 149820 299130 496650 959400 149970 299940 499900 999800 149985 299970 499900 999800 MCMO [M ] 149985 299970 499950 999900 146865 289080 475400 928800 149985 299970 499950 999900 149850 299190 496750 981400 150000 300000 500000 1000000 149985 299970 499950 999900 Table 3.4.1: Main results from the simulations at 1 Myr. 3.4. Summary of the main results ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 MMMO [M ] 10650 25470 59300 168800 10815 25920 51000 130100 30 10530 30600 91400 4200 12810 27600 77500 30 60 100 200 0 0 50 200 49 Nesc 1 1 1 1 1077 1397 1709 1990 1 1 1 1 467 675 858 1048 1 1 1 1 3 6 13 24 MPSC [M ] 139335 274500 440650 831100 123030 232170 363550 670900 149955 289440 469350 908500 138795 266940 429500 817700 149955 299910 499850 999700 149940 299790 499300 997400 MCMO [M ] 149985 299970 499950 999900 133845 258090 414550 801000 149985 299970 499950 999900 142995 279750 457100 895200 149985 299970 499950 999900 149940 299790 499350 997600 Table 3.4.2: Main results from the simulations at 5 Myr. 50 3.4. Summary of the main results ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 MMMO [M ] 11385 26190 59600 171400 12195 28800 55900 138200 2775 13920 33700 96100 5895 16200 33650 89900 30 60 100 200 0 0 100 200 Nesc 1 1 1 1 1633 1981 2304 2672 1 1 1 1 826 1078 1326 1604 1 1 1 1 13 27 55 132 MPSC [M ] 138600 273780 440350 828500 113310 211770 328900 594600 147210 286050 466250 903800 131715 251460 400050 749700 149955 299910 499850 999700 149790 299160 497150 986600 MCMO [M ] 149985 299970 499950 999900 125505 240570 384800 732800 149985 299970 499950 999900 137610 267660 433700 839600 149985 299970 499950 999900 149790 299160 497250 986800 Table 3.4.3: Main results from the simulations at 10 Myr. Chapter 4. Discussion 51 Chapter 4 Discussion This research provides support for the hypothesis that the scenario from Reinoso et al. (2020) works in Pop. III clusters above the critical mass limit. Nonetheless, there are several caveats, as we currently do not even know the mass range and the typical mass-radius relation for Pop. III clusters, so that some very important questions are open. Also the initial mass function of the Pop. III stars is unclear, the binary fraction, and other factors. It is important to acknowledge these uncertainties more specifically. Even though we do not know exactly what the radii of these Pop. III star clusters would be, we know from the study of parameters that we did that the smaller the radius of this cluster is, the more massive will be the object that will form in its center and therefore the efficiencies will be much higher. This leads us to the fact that Pop. III star clusters more massive than 1 × 106 M would have efficiencies greater than 20 % to form massive objects in their center. It should be noted that the processes of formation of structures in the universe are much more complex than just including the dynamics of bodies within a system, but it is already a good start to include more physical processes that interact with our main system in the future and check those only collisions have a high contribution in the formation process. The formation of structures in the early universe was also shaped by processes such as accretion, fragmentation, rotation, initial mass function, and the reionization of hydrogen, among others that should be considered for a complete analysis of the formation of black holes. As detailed above we take a simplified case of stellar clusters where we do not consider stellar evolution and all the stars in the cluster have the same mass and radius. The background potential that we 52 included in some of our simulations helped us to understand that although we would be modeling the effects of the gas within the cluster, it would not have a greater effect on the total mass of the most massive object at the same time, since as previously studied by Reinoso et al. (2020) simulations with a background potential could double the total mass of the most massive objects but in a much longer time. It would make sense that for denser simulations than those carried out in this investigation, they would be studied up to 1 Myr since by then they would have already formed a most massive object in their center since simulations that we made less dense for that time had not yet reached the collapse of the cluster. In the end, this is what we seek to answer as such massive structures were formed in such a short time. From the figure 3.2.3 clusters in the left side of the solid lines fulfill the condition tcoll ≤ tH or trelax ≤ tH . Clusters at 0.5 pc are in the stable region (trelax ≤ tcoll ) and the clusters at 0.1 pc and 0.05 pc, although they are in the stability region, are almost on the edge of the instability region, where collisions are increasingly greater crossing the unstable line (tcoll ≤ trelax ). As mentioned in (Escala, 2021) there is a mass limit of ∼ 4 × 108 M where all the mass turns into a central massive object. It is to be expected that simulations with masses of Pop. III star clusters similar to the latter and bigger would allow us to see this jump in efficiencies between 0 and 1. For now, with the results already obtained, we can only see how the efficiency starts at 0 and slowly these efficiencies increase. Bearing this in mind, our results are in agreement with previous works and follow a very similar line. On the other hand, we only know their characteristics theoretically about population III stars, so we will have to wait to corroborate this information in the future, which seems quite promising. In the future, we will have access to supercomputers that allow us to make much more massive and compact simulations, which will help us to analyze the collisions within these systems to calculate how these efficiencies increase. This together with other works that are being done in parallel will give us access to larger samples to be able to analyze our graphs to have a considerable sample and achieve more general conclusions. This added to what the promising images of the James Webb Space Telescope (JWST) can say in the observational counterpart and of course future space telescopes that allow us to observe the early universe such as some of them: Transient High-Energy Sky and Early Universe Surveyor (THESEUS), JWST, 53 Extremely Large Telescope (ELT), Square Kilometre Array (SKA), Wide-Field Infrared Survey Telescope (WFIRST), EUCLID, European Extremely Large Telescope (E-ELT), Giant Magellan Telescope (GMT), Thirty Meter Telescope (TMT) and Laser Interferometer Space Antenna (LISA) among others, which has us waiting for what we can observe. 54 Chapter 5. Conclusion Chapter 5 Conclusion In this investigation, therefore, support has been shown with numerical methods of a new path for the formation of massive black holes theoretically proposed by (Escala, 2021) to those previously proposed (Rees, 1984) and (Volonteri, 2010). Our sample of simulations is a simplified case of star clusters of equal mass and radius where half of our simulations have an external background potential with a plummer profile (Plummer, 1911) and the other do not. Some of these simulations after the simulation started to show an instability that causes a massive central object to form in the center of these clusters through collisions. This is called the coexistence of nuclear star clusters and black holes and it has been proposed that they formed in the same way but that they have different evolutionary mechanisms. Our systems, being located in the early universe, have masses and radii of population III stars, which are characterized by being very massive. The characteristics of these stars were taken from (Windhorst et al., 2018). We do not include stellar evolution in our calculations since our aim here is to investigate dynamical processes taking place before even the most massive main-sequence stars in the cluster have evolved. The simulations were made using NBODY6++ to study the dynamics of N bodies with an extension for parallel computers designed by (Spurzem, 1999). Our main results indicate that, on the one hand, the efficiencies we obtained are not so high due to the mass-radius relationship used for the stars. Even though Population III stars are very massive, the radii used are small, which means that in the dynamics of the stars in the cluster they have a smaller cross-section than if they had larger radii and the cross-section had a greater range of stars moving within the cluster which would cause greater 55 collisions, a more massive central object and therefore greater efficiencies. And on the other hand, all our simulations with external potential have a delay that had already been studied by (Reinoso et al., 2020) but now we consider characteristic parameters of population III stars, this means that the kinetic energy of the system is higher than when we do not consider this potential and the collapse of the cluster takes time to occur in addition to this the system is more compact and very few stars escape from the cluster. Research has recently been carried out that also explores this new scenario for the formation of black holes such as those of (Vergara et al., 2022b) and (Vergara et al., 2022a) finding that the most massive objects reach masses of approximately 104 − 105 M and the highest black hole formation efficiency is almost 50 % of the stellar mass at the end of the simulation. Three important uncertainty to consider are: first, to be able to use the initial mass function (IMF) of primordial stars. This type of work has been studied by (Hartwig et al., 2015), in which it is detailed that they constrain the lower-mass limit of the (Pop III) IMF with the total number of stars in large, unbiased surveys of the Milky Way and they conclude that the lower mass IMF limit is 0.65 M with a confidence of 95 % and that the Pop III star formation rate peaks at z ∼ 20 and that each successfully Pop III forming halo has an average stellar mass content of ∼ 100 M . It would be interesting to be able to add to our simulations the IMF of the stars of Pop. III to be able to analyze how our results vary in these cases. Another uncertainty to consider in the future is the rotation of Pop. III star clusters. In studies of (Vergara et al., 2021) it was found that rotation seems to affect the collision rate by at most 20 % and that rotation helps to retain more stars in the system, reducing the number of escapers by a factor of 2 − 3 depending on the model and the specific realization. The third uncertainty is if still gas would be present in these clusters and how it affects the evolution. This has been studied in (Boekholt et al., 2018) where it is demonstrated that the interplay between stellar dynamics, gas accretion, and protostellar evolution is particularly relevant increasing collisions from 0.1-1% of the initial population to about 10% when compared to gas-free models. This is because gas accretion onto the protostars enhances their radii, which causes an enhanced collisional cross section eventually causing major collisions inside the cluster. With the first images of a black hole so far (Event Horizon Telescope Collaboration et al., 2019) and (Collaboration et al., 2022) we are expectant to see what future 56 generations of telescopes have to show us in the coming years such as the SKA, WFIRST, EUCLID, JWST, and large ground-based facilities such as E-ELTs, GMT, and TMT (Dijkstra, 2019) among others as THESEUS or detections of other ways to study the early universe like the one found in (Bowman et al., 2018) where they report the detection of a flattened absorption profile in the sky-averaged radio spectrum, which is centered at a frequency of 78 megahertz and has a best-fitting full- width at half-maximum of 19 megahertz and an amplitude of 0.5 kelvin to study the early universe. 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https://openalex.org/W2796377982	https://europepmc.org/articles/pmc6436441?pdf=render	English	null	Thermoelectric Heat Exchange and Growth Regulation in a Continuous Yeast Culture	bioRxiv (Cold Spring Harbor Laboratory)	2,018	cc-by	3,214	1 \| INTRODUCTION generated metabolic heat by using a thermoelectric cell (Rodríguez- Barreiro, Abendroth, Vilanova, Moya, & Porcar, 2013). Nevertheless, a range of industrial fermentations are carried out in continuous cul- ture, where stable cellular densities can be maintained during long periods thanks to the supply of fresh medium, which is introduced at a rate that is equal to the volume of product that is removed from the fermenter. In this work, we aimed at designing, constructing, and characterizing a continuous culture system in which temperature is automatically controlled and electric power is constantly obtained during all the fermentation process. To do that, we envisaged, con- structed, and set in place a thermoelectric heat exchanger (hereafter called TEHE), a device also based on the Seebeck effect, which fa- cilitates a fine control of temperature and fresh medium input—and thus microbial growth—while electric power is produced. A range of parameters such as temperature, pH, or substrate con- centration need to be stable in order to sustain a suitable micro- bial growth and/or a stable biosynthesis of a bioproduct (Walker, 2000). Temperature strongly affects a range of fundamental cel- lular processes (Goldberg, 2003; Haas, 2010), and thus keeping a microbial culture in a suitable range of temperatures is of high im- portance in terms of strain performance (Amillastre, Aceves-Lara, Uribelarrea, Alfenore, & Guillouet, 2012). Large-scale growth of most microorganisms is accompanied by the production of heat (Brettel, Lamprecht, & Schaarschmidt, 1981), which, when large cul- ture volumes are set, often results in an undesirable increase in the temperature of the batch culture that has to be alleviated through refrigeration (von Stockar & van der Wielen, 1997; Türker, 2004). In a previous work, we described the first microbial thermoelec- tric cell (MTC), a system designed for batch cultures that allows the partial conversion of microbial metabolic heat into electricity. MTC is based on the Seebeck effect, a thermoelectric property that allows direct conversion of temperature differences to electricity voltage. Taking into account that microbial growth is mainly exothermic, theoretically it is possible to produce an electrical current with the Received: 6 February 2018 \| Revised: 23 March 2018 \| Accepted: 7 April 2018 Received: 6 February 2018 \| Revised: 23 March 2018 \| Accepted: 7 April 2018 Received: 6 February 2018 \| Revised: 23 March 2018 \| Accepted: 7 April 2018 DOI: 10.1002/mbo3.648 DOI: 10.1002/mbo3.648 K E Y W O R D S Funding information University of Valencia, Grant/Award Number: CPI-13-128 Funding information University of Valencia, Grant/Award Number: CPI-13-128 continuous culture, heat exchange, Peltier–Seebeck effect, power production, temperature regulation Abstract Abstract We have designed a thermoelectric heat exchanger (TEHE) for microbial fermenta- tions that is able to produce electric power from a microbial continuous culture using the intrinsic heat generated by microbial growth. While the TEHE was connected, the system proved able to stably self-maintain both the temperature and the optical den- sity of the culture. This paves the way toward a more sustainable operation of micro- bial fermentations, in which energy could be saved by converting part of the metabolic heat into usable electric power. 2Biotechnology and Synthetic Biology Laboratory, I2SysBio (Institute for Integrative Systems Biology), University of Valencia-CSIC, Paterna, Spain 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. Thermoelectric heat exchange and growth regulation in a continuous yeast culture Adriel Latorre-Pérez1 \| Cristina Vilanova1 \| José J. Alcaina2 \| Manuel Porcar1,2 © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. Correspondence Manuel Porcar, Biotechnology and Synthetic Biology Laboratory, I2SysBio (Institute for Integrative Systems Biology), University of Valencia-CSIC, Paterna, Spain. Email: manuel.porcar@uv.es Adriel Latorre-Pérez1 \| Cristina Vilanova1 \| José J. Alcaina2 \| Manuel Porcar1,2 Adriel Latorre-Pérez1 1Darwin Bioprospecting Excellence SL, Paterna, Spain MicrobiologyOpen. 2019;8:e00648. \| 1 of 5 https://doi.org/10.1002/mbo3.648 www.MicrobiologyOpen.com 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. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. \| 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. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. 2.3 The global thermal resistance (Rg) (K/W) and the whole heat capac- ity (m·cp) (J/K) of the fermenter were estimated from Equation 1: FI G U R E 1 (A) Schematic FI G U R E 1 (A) Schematic representation of the continuous culture system. (a) Thermally insulated fermenter; (b,c) peristaltic pumps; (d) Thermoelectric heat exchanger (TEHE); (e) ice bath for the compensation of temperature measurements; (f) refrigerated fresh medium tank; (g) wasted medium tank; (h) data logger; and (i) PC with a software (Rodríguez-Barreiro et al., 2013) for data recording and automatic control of the peristaltic pumps. All temperature measurements were performed with T- type thermocouples. The points at which each temperature was measured are represented as T(1–7). (B) Tridimensional representation of the TEHE constructed in this work. Sizes given in mm. (C) Zoom- in detail of the aluminum pipes inserted in the TEHE, with the thermoelectric generator cells in the interphase between both pipes sucrose was set up in the laboratory as schematically represented in Figure 1A. The TEHE consisted of two aluminum pipes of squared section and a serial connection of 10 Peltier coolers (MCPE-071- 10-13, Multicomp) placed in direct contact with the pipes. The whole device was thermally insulated with expanded polystyrene and polyurethane foam spray (Silicex Fischer, Fisher Ibérica, Tarragona, Spain) (Figure 1B). The TEHE was coupled to a thermally isolated 40 L Dewar flask (Scharlab, Barcelona, Spain) combined with a MM- 1000 overhead anchor stirrer (Labnet International, Edison, NJ), and two peristaltic pumps (Lambda Laboratory Instruments, Baar, Switzerland), which were programmed to control the flow of fresh and wasted medium. et al., 2013), the connections between the thermocouples and the data logger were performed on an ice-water mixture, which was used only as a thermal reference, to take into account the unwanted electric voltage background due to the junction of dissimilar met- als in the thermocouple data logger connection. Temperature and electrical current records were taken every 6 min throughout the experiment. Both feed and effluent flows were automatically modu- lated during all the experiment with the LabVIEW control software. 2.1 \| Experimental set-up A medium-scale continuous culture of budding yeast Saccharomyces cerevisiae strain D170 (kindly provided by Prof. Emilia Matallana, IATA, Valencia, Spain) in YPD medium supplemented with 18% \| 1 of 5 www.MicrobiologyOpen.com MicrobiologyOpen. 2019;8:e00648. https://doi.org/10.1002/mbo3.648 2 of 5 \| LATORRE-PÉREZ et al. 2 of 5 \| 2 of 5 2.2 \| Data acquisition, monitoring and recording (1) m⋅cp ⋅dTce dt = Q⋅휌⋅cp ⋅ ( Tfs−Tce ) + Pc −Tce−Tenv Rg (1) The whole system was connected to a PC in order to record tem- perature values as well as output electrical current. Temperature measurements were performed by thin T-type thermocouples in- serted into the different parts of the system and connected to a PC through a data logger. As in previous studies (Rodríguez-Barreiro where m, cp, ρ, and Tce are broth mass (kg), specific heat (J/kg·K), density (kg/m3), and temperature of the hot inlet flow (K), respec- tively, whereas Q is the flow rate (m3/s) and Pc is the metabolic heat produced by yeasts (W). Tfs (K) and Tenv (K) correspond to LATORRE-PÉREZ et al. 3 of 5 3 \| RESULTS AND DISCUSSION 3 The evolution of the yeast culture was studied in a typical ex- periment where optical density (OD) at 600 nm was periodically measured. As shown in Figure 3, yeast population and temperature during the first part of the experiment exhibited a similar pattern. When the TEHE was connected and temperature was kept constant, the OD600 of the broth was relatively stable at around eight, indicat- ing that the number of cells present in the fermenter was maintained stable despite the large flow of broth removal (2.4 L/hr on average). The output of a typical experiment carried out in the continuous culture system set as described above is shown in Figure 2. The broth (35 L) was inoculated with 700 ml (1:50) of an overnight yeast culture, and cultivated in the thermally isolated flask under shaking (180 rpm). Broth temperature rose in an exponential fashion and reached 35°C after 24 hr, (Figure 2a). At this point, culture temperature was kept constant by means of introducing fresh, cool medium in the fermenter (feed flow) at the same rate that wasted (warm) medium was extracted (effluent flow), in such a way that the volume of the culture did not change during the ex- periment. The heat flowing from the warm side to the cold side of the TEHE heated the fresh medium (from 18 up to 22°C, approxi- mately) prior to its entrance to the fermenter (Figure 2b); and, re- ciprocally, cooled the warm waste medium (with a temperature at the TEHE input of around 30°C) down to around 25°C, approxi- mately. As a result, a rather stable voltage of 1–1.3 V was recorded (Figure 2c). In order for the TEHE to produce the maximum electric power, a load resistance of 120 Ω was coupled to the terminals of the thermoelectric generators yielding 10–12 mW. When the feed and effluent flows were halted and the TEHE was not used, the temperature of the broth started rising immediately, peaked at 42°C, and then started to drop (Figure 2a). Taken together, our results prove the ability of this TEHE- based system to regulate the broth temperature and to produce electric power by harvesting metabolic heat. In our prototype, au- tonomous heating of the culture was achieved and reached values (42°C) well beyond optimal temperatures for budding yeast. fermenter, an identification assay was performed to estimate Rg and m·cp, obtaining values of 5.92 K/W and 146,547 kJ/K, respectively. The U·A constant was also estimated as explained in the Materials and Methods section, yielding a value of 1,039 W/K. where ḿ is the inlet mass flow rate (mass of water entering the TEHE per unit of time), and cp is the specific heat of water (J/kg·K). the fresh medium entering the fermenter and room temperatures, respectively. Tce and Tfe being the temperature of the hot and cold inlet flows, respectively; and Tcs and Tfs, the temperature of the hot and cold outlet flows, respectively. The q term (W) is the heat flow between the hot and the cold pipe, and it depends on the temperature of the fluids entering the exchanger. U is the global heat transmission coef- ficient (W/m3), and A (m3) is the heat exchange surface. Equations 2 and 3, describing a logarithmic mean temperature difference (LMTD) model of a heat exchanger (Mizutani, Pessoa, Queiroz, Hauan, & Grossmann, 2003), were used to mathematically characterize the TEHE: In order to obtain an estimation of the U·A constant, q was first calculated from Equation 4 in an experiment where two water flows at known temperatures were inserted in the TEHE. (2) LMTD = ΔTln = (Tce −Tfs)−(Tcs−Tfe) ln ( Tce −Tfs Tcs −Tfe ) (3) q = U⋅A⋅LMTD (2) LMTD = ΔTln = (Tce −Tfs)−(Tcs−Tfe) ln ( Tce −Tfs Tcs −Tfe ) (2) (4) q = ́m⋅cp ⋅(Tce−Tcs) = ́m⋅cp ⋅(Tfs−Tfe) (3) a) Evolution of broth (black line) and room temperatures (gray line) in a typical experiment. Arrows indicate the pe moelectric heat exchanger (TEHE) was connected. (b) Changes in the temperature of inlet and outlet flows of the ne) and power (gray line) production in the TEHE 19 24 29 34 39 44 0 20 40 60 80 100 120 Temperature (ºC) Time (hr) 15 20 25 30 35 40 21 23 25 27 29 Temperature (ºC) Time (hr) Culture Effluent flow inlet Effluent flow outlet Feed flow outlet Feed flow inlet 0 2 4 6 8 10 12 14 16 0 0,2 0,4 0,6 0,8 1 1,2 1,4 21 22 23 24 25 25 26 27 28 Power (mW) Voltage (V) Time (hr) (a) (b) (c) Culture Effluent flow inlet Effluent flow outlet Feed flow outlet Feed flow inlet Power (mW) FI G U R E 2 (a) Evolution of broth (black line) and room temperatures (gray line) in a typical experiment. Arrows indicate the period of time when the thermoelectric heat exchanger (TEHE) was connected. (b) Changes in the temperature of inlet and outlet flows of the TEHE. (c) Voltage (black line) and power (gray line) production in the TEHE 4 of 5 LATORRE-PÉREZ et al. 3 \| RESULTS AND DISCUSSION Lower, industrially friendly temperatures could be constantly maintained by means of an automatic equilibrium between the flow of fresh and product-containing media through the TEHE. This resulted in the production of a significant electric power during all the pro- cess. In addition, biomass concentration proved to be constant when the temperature was controlled. This is of key importance, since industrial bioprocesses require stable temperatures in order to maintain a constant output of a given product (Park, Lee, Chang, & Chang, 1999). The generation of electric power in heat exchangers through the Peltier–Seebeck effect has been previously reported (Bell, 2008; Riffat & Ma, 2003; Takahashi et al., 2013). However, this is, to the best of our knowledge, the first time that a heat exchanger with thermoelectric generating ability has been coupled to a bi- oprocess. The difference of temperatures that can be achieved The thermal behavior of the two main components of the system (the fermenter and the TEHE) was experimentally characterized and mathematically modeled. Following a simplified experimental set up where no fresh medium (Q=0) nor cells (Pc=0) were introduced in the FI G U R E 3 Evolution of broth temperature (black line) and optical density at 600 nm (gray squares). Arrows indicate the interval of time when the thermoelectric heat exchanger was connected. Error bars show the standard deviation of three independent measurements FI G U R E 3 Evolution of broth temperature (black line) and optical density at 600 nm (gray squares). Arrows indicate the interval of time when the thermoelectric heat exchanger was connected. Error bars show the standard deviation of three independent measurements LATORRE-PÉREZ et al. 5 of 5 between both sides of the thermoelectric generators is obviously limited by the narrow range of temperatures mesophiles can tol- erate and, therefore, the electric power that can be produced is lower than that of other type of industrial processes (Weng & Huang, 2013). Albeit low, TEHE-based power production proved to be two times more efficient compared to our previous MTC de- signed for batch culture: 255 μW were obtained from a 1.7 L yeast culture with the MTC, whereas TEHE produced up to 12 mW from a 35-L culture. Thus, a 40-fold increase in electrical power pro- duction was obtained in the TEHE compared to MTC, which was 20-fold smaller (Rodríguez-Barreiro et al., 2013). REFERENCES Amillastre, E., Aceves-Lara, C., Uribelarrea, J., Alfenore, S., & Guillouet, S. (2012). Dynamic model of temperature impact on cell viability and major product formation during fed-batch and continuous ethanolic fermentation in Saccharomyces cerevisiae. Bioresource Technology, 117, 242–250. https://doi.org/10.1016/j. biortech.2012.04.013 Bell, L. (2008). Cooling, heating, generating power, and recovering waste heat with thermoelectric systems. Science, 321(5895), 1457–1461. https://doi.org/10.1126/science.1158899 Brettel, R., Lamprecht, I., & Schaarschmidt, B. (1981). Microcalorimetric investigations of the metabolism of yeasts. Growth in batch and chemostat cultures on ethanol medium. European Journal of Applied Microbiology and Biotechnology, 11(4), 212–215. https://doi. org/10.1007/BF00505869 Goldberg, A. (2003). Protein degradation and protection against mis- folded or damaged proteins. Nature, 426(6968), 895–899. https://doi. org/10.1038/nature02263 Haas, A. (2010). Regulating the regulator: Rsp5 ubiquitinates the pro- teasome. Molecular Cell, 38(5), 623–624. https://doi.org/10.1016/j. molcel.2010.05.029 Our results are the first step toward thermoelectric power pro- duction coupled to industrial bioprocesses. The electric power ob- tained under our experimental set up was only a small percentage of the energy consumed to stir and pump the culture. However, it has to be noted that significant power production could be achieved by scaling up fermentation volumes, which might allow the design of a TEHE with an increased heat interchange surface, and more ther- moelectric generators. Also, the TEHE design could be adapted to large-scale batch fermentations, by implementing it in the physical interphase between the fermenter itself and its cooling jacket, where a significant temperature gradient takes place. This might allow an, at least, partial self-control of the culture based on its temperature, with the TEHE contributing to power the stirrer or the peristaltic pumps that regulate broth renovation rates. Mizutani, F., Pessoa, F., Queiroz, E., Hauan, S., & Grossmann, I. (2003). Mathematical programming model for heat-exchanger network syn- thesis including detailed heat-exchanger designs. 1. Shell-and-tube heat-exchanger design. Industrial and Engineering Chemistry Research, 42(17), 4009–4018. https://doi.org/10.1021/ie020964u Park, B., Lee, W., Chang, Y., & Chang, H. (1999). Long-term operation of continuous high cell density culture of Saccharomyces cerevisiae with membrane filtration and on-line cell concentration monitoring. Bioprocess Engineering, 21(2), 97–100. Riffat, S., & Ma, X. (2003). Thermoelectrics: A review of present and potential applications. Applied Thermal Engineering, 23(8), 913–935. https://doi.org/10.1016/S1359-4311(03)00012-7 Rodríguez-Barreiro, R., Abendroth, C., Vilanova, C., Moya, A., & Porcar, M. (2013). Towards a microbial thermoelectric cell. PLoS One, 8(2), Rodríguez-Barreiro, R., Abendroth, C., Vilanova, C., Moya, A., & Porcar, M. (2013). Towards a microbial thermoelectric cell. PLoS One, 8(2), e56358. 3 \| RESULTS AND DISCUSSION The metabolic heat produced by yeast growth was calculated from Equation 1, using the estimated values for Rg and cp. According to our data, 24.36 W of metabolic heat were produced under our experimental conditions. Taking into account the power production obtained in the TEHE, we estimated that the heat conversion efficiency in our system was around 0.05%. ACKNOWLEDGMENTS Türker, M. (2004). Development of biocalorimetry as a technique for process monitoring and control in technical scale fermentations. Thermochimica Acta, 419(1–2), 73–81. https://doi.org/10.1016/j. tca.2004.01.036 We are very grateful to Julián Heredero, from ICMUV (Institut Ciència Materials Universitat València) for manufacturing the TEHE pipes. This work was funded by the “Valoritza i Transfereix” pro- gram (CPI-13-128) from the University of Valencia. The authors have prepared the patent and the registration in collaboration with the Research Transfer Office (OTRI) of the University of Valencia (con- tact person, Marta Garcés: marta.garces@uv.es). We are also grate- ful to Kristie Tanner for language revision. von Stockar, U., & van der Wielen, L. (1997). Thermodynamics in bio- chemical engineering. Journal of Biotechnology, 59(1–2), 25–37. https://doi.org/10.1016/S0168-1656(97)00167-3 Walker, G. (2000). Yeast physiology and biotechnology. Chichester: John Wiley & Sons. Weng, C., & Huang, M. (2013). A simulation study of automotive waste heat recovery using a thermoelectric power generator. International Journal of Thermal Sciences, 71, 302–309. https://doi.org/10.1016/j. ijthermalsci.2013.04.008 REFERENCES https://doi.org/10.1371/journal.pone.0056358 Takahashi, K., Kanno, T., Sakai, A., Tamaki, H., Kusada, H., & Yamada, Y. (2013). Bifunctional thermoelectric tube made of tilted multilayer material as an alternative to standard heat exchangers. Scientific Reports, 3, 1501. https://doi.org/10.1038/srep01501 CONFLICT OF INTEREST The technology described in this work is subjected to industrial protection (Patent Cooperation Treaty reference number: PCT/ ES2013/000212). How to cite this article: Latorre-Pérez A, Vilanova C, Alcaina JJ, Porcar M. Thermoelectric heat exchange and growth regulation in a continuous yeast culture. MicrobiologyOpen. 2019;8:e648. https://doi.org/10.1002/mbo3.648 Adriel Latorre-Pérez http://orcid.org/0000-0002-2868-9562 ORCID Adriel Latorre-Pérez http://orcid.org/0000-0002-2868-9562
https://openalex.org/W2254428784	https://europepmc.org/articles/pmc4753342?pdf=render	English	null	Oxidative Stress in Metabolic Disorders: Pathogenesis, Prevention, and Therapeutics	Oxidative medicine and cellular longevity	2,016	cc-by	2,125	Umesh C. S. Yadav,1 Vibha Rani,2 Gagan Deep,3 Rakesh K. Singh,4 and Komara 1School of Life Sciences, Central University of Gujarat, Sector 30, Gandhinagar 382030, India Department of Biotechnology, JayPee Institute of Information Technology, A-10, Sector 62, Noida 201 307, India kaggs School of Pharmacy and Pharmaceutical Sciences University of Colorado Denver 12850 E Montview Boule 2Department of Biotechnology, JayPee Institute of Information Technology, A-10, Sector 62, Noida 201 307, India 3Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Denver, 12850 E. Montview Bou CO 80045 USA Department of Biotechnology, JayPee Institute of Information Technology, A-10, Sector 62, Noida 201 307, India 3Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Denver, 12850 E. Montview Boulevard, Aurora CO 80045, USA 4Translational Science Laboratory, College of Medicine, Florida State University, 1115 West Call St., Tallahassee, FL 32306-4300, USA 5Department of Oncologic Sciences, USA Mitchell Cancer Institute, 1660 Spring Hill Avenue, Mobile, AL 36604, USA Correspondence should be addressed to Umesh C. S. Yadav; umeshyadav@cug.ac.in Correspondence should be addressed to Umesh C. S. Yadav; umeshyadav@cug.ac.in Received 26 November 2015; Accepted 26 November 2015 Copyright © 2016 Umesh C. S. Yadav 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. Copyright © 2016 Umesh C. S. Yadav 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. Metabolic disorder is characterized by dyslipidemia, insulin refractoriness, defective insulin secretion, glucose intoler- ance, and chronic inflammation which contribute to dys- functional cellular physiology and cellular redox imbalance. The increased oxidative environment contributes to a chronic inflammatory condition in the body and creates primary risk factor for the development of several diseases such as dia- betes, arthritis, cancer, and cardiovascular complications. The increased oxidative stress in obesity and metabolic disorder can in turn cause progression and sustenance of inflamma- tory condition by upregulating redox signaling pathways and altered gene expression of inflammatory markers creating a vicious cycle. Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity Volume 2016, Article ID 9137629, 3 pages http://dx.doi.org/10.1155/2016/9137629 Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity Volume 2016, Article ID 9137629, 3 pages http://dx.doi.org/10.1155/2016/9137629 Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity Volume 2016, Article ID 9137629, 3 pages http://dx.doi.org/10.1155/2016/9137629 Editorial Oxidative Stress in Metabolic Disorders: Pathogenesis, Prevention, and Therapeutics Umesh C. S. Yadav,1 Vibha Rani,2 Gagan Deep,3 Rakesh K. Singh,4 and Komaraiah Palle5 1School of Life Sciences, Central University of Gujarat, Sector 30, Gandhinagar 382030, India 2Department of Biotechnology, JayPee Institute of Information Technology, A-10, Sector 62, Noida 201 307, India 3Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Denver, 12850 E. Montview Boulevard, Aurora, CO 80045, USA 4 Umesh C. S. Yadav,1 Vibha Rani,2 Gagan Deep,3 Rakesh K. Singh,4 and Komara Addition- ally, the continued intensive research in this area is yielding In the obese diabetic patients, excessive uric acid has been shown to induce cardiovascular disease (CVD) through the Oxidative Medicine and Cellular Longevity 2 fibrotic diseases, and inflammatory pathways, the authors concluded that more investigations are warranted to clarify its role in high fat diet-induced obesity and diabetes. generation of ROS and subsequent endothelial dysfunction [5]. The epidemiological studies suggest that uric acid is an independent risk factor for developing CVDs, especially in people with diabetes, hypertension, and heart failure [6]. However, the clear molecular targets of uric acids have not yet been identified. In this issue, H. Xie et al. presented that uric acid induces ROS generation and increased the expression of several inflammatory molecules such as IL-6, TNF-alpha, and MCP-1 through Notch-1 pathway. Silencing of Notch- 1 reversed these changes, and so did (−)-epigallocatechin- 3-gallate (EGCG) treatment via inhibiting Notch-1. Authors concluded that downregulation of Notch-1 by EGCG could be an effective approach to decrease inflammation and oxidative stress induced by uric acid. While presenting association between oxidative stress marker plasma 8-isoprostane and the activity of paraoxonase- 1 with coronary artery disease (CAD), A. Kuchta et al. sug- gested that 8-isoprostane could be an important biomarker of lipid peroxidation. They reported a correlation of decreased activity of paraoxonase-1 with decreased protection against lipid oxidation which could be linked with CAD. High Mobility Group Box-1 (HMGB-1) is a nuclear protein that regulates the transcription of many genes and also involved in the immune response and inflammation. H. Wu et al. investigated how HMGB-1 protein is associated with diabetes-induced oxidative stress that critically regulates endothelial progenitor cell (EPC) dysfunction. In their study, they showed that advanced glycation end products enhanced HMGB-1 levels in EPCs which was downregulated by antioxi- dant N-acetylcysteine (NAC), suggesting that HMGB-1 could be an important regulator in diabetes-induced oxidative stress and resultant EPC dysfunction. In their study, K. Gawlik et al. explored the antioxidant defense biomarkers present in the blood of type-2 diabetes patients. They reported that ferric reducing antioxidant power (FRAP) and uric acid levels were significantly elevated in obese diabetic patients; however, no significant difference was observed in the biomarkers of antioxidant defense system between patients with or without chronic diabetes. Umesh C. S. Yadav,1 Vibha Rani,2 Gagan Deep,3 Rakesh K. Singh,4 and Komara They concluded that in the presence of excessive ROS production the antioxidant system tends to crumble as reflected by decreased level of erythrocytes glutathione. A. Keller et al. discussed the critical role of mitochondrial perturbation in vascular smooth muscle cells caused by nutritional stress during diabetes. They explored the premise that nutritional stress in diabetes may impair adaptive mitochondrial plasticity via NOS-mediated pathway. They reported that in Goto-Kakizaki diabetes mouse model high glucose enhanced nitric oxide, ROS, and respiratory control ratio while at the same time decreasing phospho-eNOS, uncoupled respiration and mitochondrial respiratory com- plex expression. With these findings, the authors suggested that eNOS and mitochondria could be potential drug targets in nutritional stress-induced vascular pathogenesis.i Y. Wang et al. discussed the role of peptidyl-prolyl isomerase (Pin-1), a key protein involved in cell division and many other cellular functions both in yeast and humans, in protection against high-dose alcohol-induced apoptosis in mouse cardiomyocytes. They reported that, on the one hand, high-dose alcohol-induced Pin-1 promotes mitochondrial dysfunction leading to ROS increase, while, on the other hand, Pin-1 suppresses endothelial nitric oxide synthase (eNOS) expression leading to apoptosis of cardiomyocytes. The authors concluded that due to these critical activities in alcohol-stimulated cardiomyocytes Pin-1 could be a potential therapeutic target in alcohol-induced cardiomyopathy. Overall, several new findings have been presented in this special issue which have further advanced our knowledge in this exciting area. Also, these studies suggest that oxidative stress is a critical component of metabolic disorders and sustained research efforts are necessary to unravel the com- plexity associated with oxidative stress in metabolic disorders and related pathologies. In a review article, I. Petyaev elaborated the important role of lycopene, a member of tetraterpene carotenoids, in scavenging the lipid radicals, ROS, and nitric oxide affording protective effects against prooxidant species. However, with ageing and increased accumulation of oxidative species in the body, lycopene effectiveness might be depleted leading to type-2 diabetes and increased risk for CVDs. The limited bioavailability of lycopene due to decreased intestinal absorp- tion with aging and its enzymatic and oxidative degradation could be addressed by designing novel nutraceuticals. Acknowledgment We acknowledge and thank the participating authors and reviewers in the successful compilation of this special issue. Galectin-3 (Gal-3), a member of lectin family and impor- tant regulator of cellular metabolism, has been implicated in obesity, type-2 diabetes, heart failure, and cancer [7– 11]. S. Menini et al. in their review article discussed the controversial role of Gal-3 in this field. On the one hand, Gal-3 has been found elevated in obese and diabetic patients, while, on the other hand, Gal-3 knockout mice showed increased adiposity and systemic inflammation related with altered glucose homeostasis suggesting negative modulation of nutrition-induced immune responses. On the contrary, a few studies showed decrease in fat mass and body weight in high fat diet-fed Gal-3 knockout mice. Although Gal-3 has emerged as an important prognostic marker for heart failure, Umesh C. S. Yadav Vibha Rani Gagan Deep Rakesh K. Singh Komaraiah Palle Umesh C. S. Yadav,1 Vibha Rani,2 Gagan Deep,3 Rakesh K. Singh,4 and Komara novel information which is vital towards further advancing our understanding in this complex field.f Metabolic disorder is characterized by dyslipidemia, insulin refractoriness, defective insulin secretion, glucose intoler- ance, and chronic inflammation which contribute to dys- functional cellular physiology and cellular redox imbalance.h g pi Till recently, the major focus and efforts have been towards employing antioxidant as preventive/therapeutic agent against metabolic disorders, considering the central role of oxidative stress therein. However, several studies including clinical ones have clearly shown that this strategy has not yielded expected outcomes [3, 4]. Alternatively, several studies have suggested that it would be better to target the pathways involved in reactive oxygen species (ROS) generation rather than ROS neutralization by antioxidants [1, 2]. Several studies have also shown that cells own machinery gets overinvolved in the production of ROS and resultant molecular signaling leads to activation of inflammatory sig- naling and incessant inflammation. Mitochondrial dysfunc- tion led ROS generation, weakening of cellular antioxidant machinery, aberrant activation of NADPH oxidase system, and lipid peroxidation mechanisms are few examples of oxidative stress sources. Several new molecular targets that are affected by oxidative stress and in turn distort the cellular physiology resulting in pathogenesis are reported in this special issue. These targets include NOTCH-1, Pin-1, galectin- 3, paraoxonase-1, HMGB-1, and mitochondrial respiratory complexes. The increased oxidative environment contributes to a chronic inflammatory condition in the body and creates primary risk factor for the development of several diseases such as dia- betes, arthritis, cancer, and cardiovascular complications. The increased oxidative stress in obesity and metabolic disorder can in turn cause progression and sustenance of inflamma- tory condition by upregulating redox signaling pathways and altered gene expression of inflammatory markers creating a vicious cycle. Many evidences implicate oxidative stress in the metabolic perturbations in obesity, diabetes, and cardiovas- cular pathogenesis [1, 2]. The oxidative damage and inflammation originate from both our environmental milieu such as chemicals, toxicants, and nutrients and body’s own metabolism as metabolic byproducts and intermediate. The understanding of their roles and effects on cellular physiology has led to the development of novel biomarkers and molecular targets which could be useful in devising innovative approaches in the prevention, diagnosis, and treatment of inflammatory and metabolic diseases. [1] I. Savini, M. V. Catani, D. Evangelista, V. Gasperi, and L. Avigliano, “Obesity-associated oxidative stress: strategies final- ized to improve redox state,” International Journal of Molecular Sciences, vol. 14, no. 5, pp. 10497–10538, 2013. References 3 Oxidative Medicine and Cellular Longevity Oxidative Medicine and Cellular Longevity 3 [2] P. Manna and S. K. Jain, “Obesity, oxidative stress, adipose tissue dysfunction, and the associated health risks: causes and therapeutic strategies,” Metabolic Syndrome and Related Dis- orders, vol. 13, no. 10, pp. 423–444, 2015. [3] M. P. Valdecantos, P. P´erez-Matute, and J. A. Mart´ınez, “Obesity and oxidative stress: role of antioxidant supplementation,” Revista de Investigacion Clinica, vol. 61, no. 2, pp. 127–139, 2009. [4] V. Mishra, “Oxidative stress and role of antioxidant supplemen- tation in critical illness,” Clinical Laboratory, vol. 53, no. 3-4, pp. 199–209, 2007. [5] U. M. Khosla, S. Zharikov, J. L. Finch et al., “Hyperuricemia induces endothelial dysfunction,” Kidney International, vol. 67, no. 5, pp. 1739–1742, 2005. [6] J. F. Baker, E. Krishnan, L. Chen, and H. R. Schumacher, “Serum uric acid and cardiovascular disease: recent developments, and where do they leave us?” American Journal of Medicine, vol. 118, no. 8, pp. 816–826, 2005. [7] F.-T. Liu and G. A. Rabinovich, “Galectins as modulators of tumour progression,” Nature Reviews Cancer, vol. 5, no. 1, pp. 29–41, 2005. [8] N. N. Pejnovic, J. M. Pantic, I. P. Jovanovic et al., “Galectin- 3 deficiency accelerates high-fat diet-induced obesity and amplifies inflammation in adipose tissue and pancreatic islets,” Diabetes, vol. 62, no. 6, pp. 1932–1944, 2013. [9] H. Yilmaz, M. Cakmak, O. Inan, T. Darcin, and A. Akcay, “Increased levels of galectin-3 were associated with prediabetes and diabetes: new risk factor?” Journal of Endocrinological Investigation, vol. 38, no. 5, pp. 527–533, 2015. [10] A. Chen, W. Hou, Y. Zhang, Y. Chen, and B. He, “Prognostic value of serum galectin-3 in patients with heart failure: a meta- analysis,” International Journal of Cardiology, vol. 182, pp. 168– 170, 2015. [11] N. Rubinstein, J. M. Ilarregui, M. A. Toscano, and G. A. Rabi- novich, “The role of galectins in the initiation, amplification and resolution of the inflammatory response,” Tissue Antigens, vol. 64, no. 1, pp. 1–12, 2004.
https://openalex.org/W3184169928	https://ojs.staialfurqan.ac.id/jtm/article/download/119/75	Indonesian	null	Analisis Permasalahan Transportasi di Perkotaan: Studi Kasus pada Kawasan Perkotaan Yogyakarta	Jurnal Tana Mana	2,021	cc-by-sa	2,210	Abstrak Penelitian ini bertujuan untuk menganalisis permalasahan transportasi di perkotaan studi kasus pada kawasan perkotaan yogyakarta. Adapun jenis penelitian ini adalah penelitian kualitatif dengan sumber data diperoleh dari sumber data primer dan sumber data sekunder. Data penelitian yang terkumpul kemudian dianalisis dengan pendekatan ilmu sains. Dari penelitian yang dilakukan diperoleh hasil bahwa permasalahan transportasi di perkotaan khususnya di Kawasan Perkotaan Yogyakarta disebabkan oleh yang pertama belum adanya sarana dan prasana yang memadai di Kawasan Perkotaan Yogyakarta. Kedua, rendahnya kesadaran masyarakat terhadap pemanfaatan fasilitas lalu lintas dan perlengkapan jalan. Kemudian, di Kawasan Perkotaan Yogyakarta belum mengimplementasikan dengan baik tentang manajemen kebutuhan lalu lintas untuk meningkatkan efesiensi dan efektifitas penggunaan lalu lintas dan mengendalikan pergerakan lalu lintas. Disamping itu, laju pertumbungan penduduk yang cukup tinggi di Kawasan Perkotaan Yogyakarta berakibat pada kemacetan lalu lintas di wilayah tersebut. Permasalahan transportasi yang memiliki eksternalitas lintas daerah seperti daerah Kawasan Perkotaan Yogyakarta tidak mungkin dilakukan dan ditangani oleh daerah sendiri Kata Kunci: Analisis Permasalahan Perkotaan, Studi Kasus Kawasan, Permasalahan Transportasi Kata Kunci: Analisis Permasalahan Perkotaan, Studi Kasus Kawasan, Permasalahan Transportasi Website https://ojs.staialfurqan.ac.id/jtm/index This is an open access article under the CC BY SA license https://creativecommons.org/licenses/by-sa/4.0/ Jurnal Tana Mana Vol. 2, No. 1, June 2021 https://ojs.staialfurqan.ac.id/jtm/index Analisis Permasalahan Transportasi di Perkotaan: Studi Kasus pada Kawasan Perkotaan Yogyakarta Galih Puji Kurniawan1, Salsabila Zahra Shalikhah2, Hanifah Shofiati3, Nuha Nur Azizah4, Mahmud Mochtar5 12345Universitas Gadjah Mada, Indonesia Galih Puji Kurniawan1, Salsabila Zahra Shalikhah2, Hanifah Shofiati3, Nuha Nur Azizah4, Mahmud Mochtar5 12345Universitas Gadjah Mada, Indonesia E-mail galih.p.k@mail.ugm.ac.id* PENDAHULUAN Transportasi memiliki peran strategis dalam proses pembangunan, yang tidak hanya dapat mendorong pembangunan daerah, tetapi juga mendukung pembangunan ekonomi nasional. Menurut Abbas Salim dalam bukunya Manajemen Transportasi (2002) pembangunan ekonomi membutuhkan jasa angkutan yang cukup serta memadai, tanpa adanya transportasi sebagai sarana penunjang tidak dapat diharapkan tercapainya hasil yang memuaskan dalam usaha pengembangan ekonomi dari suatu Negara (Siwu, 2021; Kadir, 2006; Junaidi, et al., 2020). Terlepas dari tingkat perkembangan ekonomi masing-masing negara, dalam menyusun sistem transportasi atau menentukan kebijakan transportasi dengan lingkup nasional, perlu ditentukan terlebih dahulu tujuan yang ingin dicapai dan sistem pelayanan transportasi apa yang dibutuhkan dalam transportasi negara tersebut. g Menurut Undang-Undang No. 22 Tahun 2009 tentang Lalu Lintas dan Angkutan Jalan disebutkan bahwa lalu lintas dan angkutan jalan sebagai bagian dari sistem transportasi nasional harus dikembangkan potensi dan perannya untuk mewujudkan keamanan, keselamatan, ketertiban, dan kelancaran berlalu lintas dan angkutan jalan dalam rangka mendukung pembangunan ekonomi dan pembangunan wilayah. Berdasarkan pendapat ahli dan undang-undang terkait transportasi, tidak dapat dipungkiri bahwa transportasi merupakan bagian terpenting dalam suatu negara, khususnya di perkotaan. Apabila suatu perkotaan transportasinya bermasalah, maka akan mengakibatkan aktivitas lainnya bermasalah. Hal ini selaras dengan pendapat Peneliti Pusat Studi Transportasi dan Logistik (PUSTRAL) UGM, Lilik Wachid Budi Susilo, yang mengutarakan bahwa persoalan transportasi perlu mendapat perhatian yang segera dan serius serta menjadi prioritas dalam pembangunan kota. Melihat pentingnya peran transportasi dalam suatu perkotaaan, maka diperlukan manajemen pengelolaan transportasi kota yang baik agar dapat mempermudah mobilitas masyarakat perkotaan dalam melakukan aktivitas. Selain itu, kelengkapan sarana dan prasarana transportasi perkotaan yang baik akan mampu memberikan pelayanan kepada masyarakat dalam melakukan mobilitas. Terdapat beragam moda transportasi diperkotaan, khususnya di Kawasan Perkotaan Yogyakarta berdasarkan data dari Kota Jogja.com daerah Kawasan Perkotaan Yogyakarta memiliki berbagai macam moda transportasi, diantaranya adalah Becak, Andong, Bis Kota, Angkot, Ojek (online dan offline), Trans Jogja, Taksi, dan para warga yang berlalu lalang menggunakan kendaraan pribadi mereka baik motor maupun mobil. Transportasi merupakan bagian integral dari proses produksi barang dan jasa yang akan memberikan kontribusi manfaat yang besar terhadap pertumbuhan ekonomi, mengurangi kemiskinan dan kesinambungan lingkungan. Efisiensi dan efektifitas transportasi menjadi parameter utama (Solikatun, S., & Masruroh, 2014; Warsilan, W., & Noor, 2015). Lalu lintas dan angkutan jalan sebagai bagian dari sistem transportasi nasional harus dikembangkan potensi dan perannya untuk mewujudkan keamanan, keselamatan, ketetiban, dan kelancaran berlalu lintas dan angkutan jalan dalam rangka mendukung pembangunan ekonomi dan pembangunan wilayah. PENDAHULUAN Namun hingga saat ini, permasalahan transportasi di wilayah perkotaan masihlah sangat kompleks dan belum tertangani secara terencana dan berkelanjutan. Oleh karena itu, perlu dikaji lebih dalam lagi mengenai permasalahan transportasi perkotaan agar menjadi masukan kepada pemangku kebijakan dalam penataan sistem transportasi perkotaan yang efisiensi dan efektif. Tujuan dari penelitian ini adalah untuk mengetahui permasalahan sarana dan prasarana transportasi di Kawasan Perkotaan Yogyakarta. gy Mengetahui pengaturan transportasi di Kawasan Perkotaan Yogyakarta dan mengetahui faktor yang menyebabkan kemacetan di Kawasan Perkotaan Yogyakarta. METODE Metode pengumpulan data penelitian ini menggunakan metode 1) library research. Teknik analisis penelitian ini menggunakan teknik analisis non statistic dengan menggunakan teknik analisa data kualitatif dengan dengan pendekatan tafsir ilmy dan metode sains modern. Metode Pengumpulan Data Dalam metode pengumpulan data ini, penulis menggunakan library research, yaitu mengumpulkan data lewat buku-buku perpustakaan atau karya ilmiah lainnya yang berkaitan dengan masalah yang diteliti. Pada penelitian kepustakaan, data atau bahan yang di peroleh adalah data sekunder yang berasal dari bahan hukum primer, bahan hukum sekunder, dan bahan hukum tertier. Metode Pengelolahan dan Analisis Data Data yang dikumpulkan, baik data primer maupun data skunder, kemudian diolah melalui metode kualitatif, yaitu pengolahan gagasan, ulasan, pandangan dari berbagai ahli tentang masalah yang berhubungan permasalahan transportasi di perkotaan. 45 HASIL DAN PEMBAHASAN 1.1 Masalah Sarana dan Prasarana Transportasi Hampir semua kota besar di Indonesia mengalami masalah transportasi yang kompleks, salah satunya adalah masalah sarana dan prasarana transportasi yang mengakibatkan kemacetan lalu lintas yang semakin parah. Pertambahan jumlah kendaraan bermotor yang tidak diimbangi dengan peningkatan infrastruktur jalan raya merupakan salah satu penyebab kemacetan. Peningkatan jumlah kendaraan terjadi seiring bertambahnya jumlah penduduk karena kendaraan dibutuhkan untuk memfasilitasi pergerakan masyarakat. Transportasi atau kendaraan merupakan kebutuhan turunan akibat adanya aktivitas ekonomi, sosial, budaya, dan sebagainya (Lestari, 2014). Tabel 1 menunjukan peningkatan jumlah kendaraan bermotor di Kawasan Perkotaan Yogyakarta pada kurun waktu 2011-2015: Tabel 1. Jumlah Peningkatan Kendaraan Bermotor di DI Yogyakarta dalam 4 Tahun Tabel 1. Jumlah Peningkatan Kendaraan Bermotor di DI Yogyakarta dalam 4 Tahun Tabel 1. Jumlah Peningkatan Kendaraan Bermotor di DI Yogyakarta dalam 4 Tahun Berdasarkan Tabel 1 dapat dilihat bahwa jumlah kendaraan bermotor di Daerah Istimewa Yogyakarta setiap tahunnya mengalami peningkatan. Menurut Asikin (2011:10) mengatakan bahwa sebagai kawasan perkotaan, jumlah kendaraan bermotor yang relatif tinggi, maka Kawasan Perkotaan Yogyakarta secara umum akan menghadapi permasalahan transportasi perkotaan sebagai berikut: 1. Pertumbuhan kebutuhan transportasi sering tidak seimbang dengan penyedia prasarana. 2. Kualitas dan jumlah angkutan umum yang belum memadai sarana dan prasarana, jaringan jalan dan jaringan trayek, terminal, sistem pengendalian dan pelayanan angkutan umum belum berhasil ditata secara konsepsional. p 3. Makin jauh jarak perjalanan harian masyarakat. 4. Penggunaan kendaraan pribadi yang kurang efisien. 5. Di daerah perkotaan timbul kemacetan, kesemrawutan, dan pencemaran lingkungan. 46 Salah satu cara untuk mengatasi kemacetan yang ada di Kawasan Perkotaan Yogyakarta adalah dengan beralih dari kendaraan pribadi ke transportasi umum. Saat ini, kondisi transportasi umum di Kawasan Perkotaan Yogyakarta sendiri bisa dibilang kurang baik. Rendahnya kualitas transportasi umum di Kawasan Perkotaan Yogyakarta ditandai dengan timbulnya masalah-masalah transportasi umum yang saling mempengaruhi satu sama lain, faktor-faktor penyebab rendahnya kualitas transportasi di Kawasan Perkotaan Yogyakarta juga disebabkan oleh berbagai faktor dan masalah lain yang cukup kompleks. Faktor-faktor penyebab rendahnya kualitas transportasi umum di Kawasan Perkotaan Yogyakarta, sebaga berikut: 1. Dana pengadaan atau peremajaan fasilitas transportasi yang tidak mencukupi. p 2. Kurangnya pengawasan dari pemerintah atau pihak yang berkewajiban. 3. Kurangnya kesadaran masyarakat untuk ikut menjaga fasilitas sarana dan prasarana transportasi. 4. Kurangnya disiplin masyarakat. Pertumbuhan dan perkembangan suatu kota yang pesat tanpa diikuti dengan pengadaan sistem transportasi yang memadai untuk ukuran kota itu merupakan bentuk besarnya permintaan daripada penawaran, begitu pula sebaliknya. HASIL DAN PEMBAHASAN Lajunya pertumbuhan sistem transportasi yang tidak sesuai dengan ukuran perkembangan suatu kota, merupakan wujud penawaran lebih besar daripada permintaan untuk transportasi. Kondisi-kondisi yang telah disebutkan di atas akan berakibat pada timbulnya permasalahan-permasalahan baru dalam sistem transportasi maupun permasalahan perkotaan pada umumnya. Jika ditinjau dari segi fisik misalnya, cukup banyak angkutan umum di Kawasan Perkotaan Yogyakarta yang rusak dan sebenarnya sudah seharusnya mendapatkan uji kelayakan. Kerangka bus yang rusak, jok yang sobek, dan mesin bus yang menghasilkan asap berlebihan tentunya akan menimbulkan kesan kotor dan semrawut sehingga ini jelas akan menurunkan daya tarik angkutan itu sendiri bagi penumpang. Belum lagi rute trayek angkutan kota yang tidak efisien sehingga mengakibatkan waktu perjalanan menjadi lebih lama. Dengan kondisi yang demikian, tentunya orang-orang akan lebih tertarik untuk menggunakan kendaraan pribadi mereka. 1.2 Peraturan Transportasi 1.2 Peraturan Transportasi Pengaturan transportasi adalah kegiatan penetapan kebijakan transportasi, lalu lintas pada jaringan atau ruas-ruas jalan tertentu. Pengaturan transportasi diperlukan untuk mengatur transportasi umum di Kawasan Perkotaan Yogyakarta, agar terpenuhi kebutuhan masyarakat akan transportasi umum. Dikarenakan pertumbuhan penduduk yang pesat maka kebutuhan akan transportasi umum juga akan bertambah dan harus dipenuhi oleh Pemerintah Yogyakarta. Transportasi umum yang sering dijumpai di Yogyakarta adalah bus trans jogja, seperti yang termuat di Pergub DIY No. 109 Tahun 2020 tentang Penugasan Pengelolaan Sistem Angkutan Perkotaan Bersubsidi Trans Jogja Dengan Sistem Buy The Service Kepada Pt. Anindya Mitra Internasional “Trans Jogja adalah sistem pengelolaan angkutan penumpang umum bersubsidi di Kawasan Perkotaan Yogyakarta yang diselenggarakan dengan sistem buy the service atau membeli pelayanan yang diselenggarakan oleh Pemerintah Daerah.” Tahun 2020 tentang Penugasan Pengelolaan Sistem Angkutan Perkotaan Bersubsidi Trans Jogja Dengan Sistem Buy The Service Kepada Pt. Anindya Mitra Internasional “Trans Jogja adalah sistem pengelolaan angkutan penumpang umum bersubsidi di Kawasan Perkotaan Yogyakarta yang diselenggarakan dengan sistem buy the service atau membeli pelayanan yang diselenggarakan oleh Pemerintah Daerah.” 47 Namun di masa pandemi ini minat masyarakat sekitar untuk menggunakan transportasi umum menurun, salah satu transportasi umum di Yogyakarta yang merasakan dampaknya adalah bus trans jogja, menurut artikel tribunnews “Jumlah penumpang angkutan umum Trans Jogja mengalami penurunan signifikan selama masa pandemi Covid-19. Dalam kondisi normal jumlah penumpang per hari mencapai 20.000 sampai 22.000 penumpang, kini rata-rata hanya 1.402 orang per hari”. Oleh karena itu pemerintah jogja menerapkan kebijakan untuk mengurangi unit trans jogja yang beroperasi di jalan. Penerapan social distancing juga dilakukan di bus trans jogja, yang biasanya satu bus trans jogja dapat diisi 40 orang di masa pandemi satu bus hanya bisa diisi 20 orang saja. 1.3 Kemacetan Lalu Lintas Kemacetan lalu lintas merupakan masalah dan telah menjadi simbol utama di kota-kota besar seperti Jakarta, Surabaya, Bandung dan Yogyakarta. Yogyakarta sendiri merupakan kota dengan arus lalu lintas yang cukup padat. Jumlah penduduk Yogyakarta yang meningkat pesat menyebabkan kemacetan lalu lintas di beberapa lokasi dan hampir semua lokasi. Menurut BPS Provinsi DIY (2016), pada tahun 2015 jumlah penduduk DIY mencapai 3.679.176 jiwa dengan kepadatan penduduk tercatat 1.555 jiwa per km. Kepadatan tertinggi terjadi di Kota Yogyakarta, mencapai 12.699 KESIMPULAN Dari uraian diatas dapat ditarik kesimpulan bahwa permasalahan transportasi di perkotaan khususnya di Kawasan Perkotaan Yogyakarta disebabkan oleh yang pertama belum adanya sarana dan prasana yang memadai di Kawasan Perkotaan Yogyakarta. Kedua, rendahnya kesadaran masyarakat terhadap pemanfaatan fasilitas lalu lintas dan perlengkapan jalan. Kemudian, di Kawasan Perkotaan Yogyakarta belum mengimplementasikan dengan baik tentang manajemen kebutuhan lalu lintas untuk meningkatkan efesiensi dan efektifitas penggunaan lalu lintas dan mengendalikan pergerakan lalu lintas. Disamping itu, laju pertumbungan penduduk yang cukup tinggi di Kawasan Perkotaan Yogyakarta berakibat pada kemacetan lalu lintas di wilayah tersebut. Permasalahan transportasi yang memiliki eksternalitas lintas daerah seperti daerah Kawasan Perkotaan Yogyakarta tidak mungkin dilakukan dan ditangani oleh daerah sendiri, diperlukan koordinasi dan kolaborasi antar daerah karena urusan tersebut akan lebih efisien dan optimal jika dikelola bersama sebagai satu sistem tanpa melihat batas wilayah administratif. ACKNOWLEDGEMENT The authors wish to thank to Dra. Nur Aini Yuniarti, M.Si. was support for this research. Acknowledgments were also extended to all who contributed to this research. Thank you also expressed to all participants for their contribution in helping collect data and in testing research REFERENSI Agustina Dewi. Mulai Hari Ini Bus Trans Jogja Hanya Beroperasi 83 Unit dari Sebelumnya 116 Armada, at: https://www.tribunnews.com/regional/2020/05/01/mulai-hari-ini-bus- trans-jogja-hanya-beroperasi-83-unit-dari-sebelumnya-116-armada?page=all [Diakses pada 20 Maret 2021 Pukul 19.00 WIB] Anonim. 2016. Transportasi di Jogja, at: https://www.google.co.id/amp/s/www.kotajogja.com/88/transportasi-di- jogja/%3famp [Diakses pada 18 Maret 2021 Pukul 15.30 WIB] 48 BPS Provinsi D.I. Yogyakarta, 2012-2016. Provinsi Daerah Istimewa Yogyakarta Dalam Angka 2012-2016. Yogyakarta: BPSD.I.Yogyakarta. g gy gy BPS Provinsi D.I. Yogyakarta, 2016. Provinsi Daerah Istimewa Yogyakarta Dalam A k 2020 Y k t BPSD I Y k t g gy gy BPS Provinsi D.I. Yogyakarta, 2016. Provinsi Daerah Istimewa Yogyakarta Dalam Angka 2020. Yogyakarta: BPSD.I.Yogyakarta. Junaidi, J., Gani, I., & Noor, A. (2020). Analisis transportasi darat terhadap pertumbuha ekonomi di provinsi kalimantan timur. KINERJA, 17(2), 264-269. Kadir, A. (2006). Transportasi: peran dan dampaknya dalam pertumbuhan ekonomi nasional. Jurnal perencanaan dan pengembangan wilayah wahana hijau, 1(3), 121- 131. Novatiani, R Ait dan Nurmalita Lestari, 2014. Pengaruh Akuntabilitas Publik, Partisipasi Masyarakat dan Transparansi Kebijakan Publik Terhadap Pengawasan Keuangan Daerah (APBD) di Kota DPRD Kota Bandung. Jurnal. ( ) g J Siwu, H. F. D. (2021). Permintaan dan Penawaran Jasa Transportasi. Jurnal Pembanguna Ekonomi Dan Keuangan Daerah, 19(2), 114-122. g ( ) Solikatun, S., & Masruroh, Y. (2014). Kemiskinan Dalam Pembangunan. Jurnal Analis Sosiologi, 3(1). Tamara, S., & Sasana, H. (2017). Analisis Dampak Ekonomi Dan Sosial Akibat Kemacetan Lalu Lintas Di Jalan Raya Bogor-Jakarta. Jurnal REP (Riset Ekonomi Pembangunan), 2(2), 185–196 Warsilan, W., & Noor, A. (2015). Peranan infrastruktur terhadap pertumbuhan ekonomi dan implikasi pada kebijakan pembangunan di kota samarinda. MIMBAR: Jurnal Sosial dan Pembangunan, 31(2), 359-366. Copyright Holder : © Galih Puji Kurniawan1*, Salsabila Zahra Shalikhah2, Hanifah Shofiati3, Nuha Nur Azizah4, Mahmud Mochtar5 First Publication Right : © Jurnal Tana Mana This article is under: CC BY SA 49 49
https://openalex.org/W2943659942	https://www.iiste.org/Journals/index.php/RHSS/article/download/47541/49087	English	null	The Effect of Parenting Styles on Strengths of Humanity: Love, Kindness and Social Intelligence in Myanmar Adolescents	Research on Humanities and Social Sciences	2,019	cc-by	8,022	www.iiste.org www.iiste.org Research on Humanities and Social Sciences Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.8, 2019 The Effect of Parenting Styles on Strengths of Humanity: Love, Kindness and Social Intelligence in Myanmar Adolescents Thae Su Mo School of Psychology, Northeast Normal University 5268 Renmin Street, Changchun 130024, China Abstract The main purpose of this study was to explore the effect of parenting styles on strengths of humanity (love, kindness and social intelligence) in Myanmar adolescents. 378 students from three selected secondary schools participated in this study. The Scale of Parenting Styles developed by Gafoor and Kurkkan (2014) was used to measure parental responsiveness and control, for identifying parenting styles. Strengths of humanity were measured through 96-item Value in Action Inventory designed by Park and Peterson (2006). The results showed that adolescents perceived their mothers as more responsive and more controlling than their father. Perceptions with parental responsiveness and control were not different between boys and girls. Maternal responsiveness positively predicted strengths of love and kindness, while paternal control positively predicted social intelligence. Adolescents of authoritative mothers had higher scores in all humanity strengths than those of negligent mothers, and higher scores of kindness than those of permissive mothers. Similarly, adolescents of authoritative fathers reported higher scores in all humanity strengths than those of negligent fathers, and higher scores of kindness than those of permissive fathers. Adolescents of negligent mothers also revealed lower level of love than those of permissive mothers, while adolescents of negligent fathers revealed lower level of love than those of authoritarian fathers. In social intelligence, adolescents of negligent fathers were lower than their counterparts of parents who practiced other three styles. Adolescents of permissive and authoritarian styles did not show significant differences in all three strengths of humanity. On the whole, authoritative parenting style was the best, and negligent style was the worst. Keywords: Parenting Style, Character Strengths, Strengths of Humanity, Love, Kindness, Social Intelligence, Myanmar DOI: 10.7176/RHSS/9-8-05 DOI: 10.7176/RHSS/9-8-05 DOI: 10.7176/RHSS/9-8-05 DOI: 10.7176/RHSS/9-8-05 Publication date: April 30th 2019 Publication date: April 30th 2019 1. Introduction P i h l Specifically, children and adolescents of authoritarian families were lower in social skills and self-esteem, and revealed higher levels of depression; those of permissive families demonstrated more involvement in problematic behaviour and lower performance in school: those of negligent homes showed 27 www.iiste.org Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.8, 2019 Research on Humanities and Social Sciences Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.8, 2019 www.iiste.org the poorest performance in all domains of competence. the poorest performance in all domains of competence. However, some studies found that authoritative parenting was not related to the school grades of Asian Americans (Dornbusch, Ritter, Leiderman, Roberts, & Fraleigh, 1987), and Hong Kong Chinese (Leung, Lau, & Lam, 1998; McBride-Chang, & Chang, 1998). In the study of “Parenting Styles of Hmong Parents and its Effects and Contributions to Hmong Student's Academic Achievement”, Moua (2010) found that although Hmong parents were more authoritarian type, the school performance of their children was better than Caucasian students of authoritative parents. Moua argued that “Diana Baumrind's parenting styles may not necessarily reflect those of other ethnic cultures” (p.3). In her study of “consequences of parenting style for Chinese Americans and European Americans”, Chao (2001) found that school performance of Chinese youths whose parents were authoritative were not better than Chinese youths of authoritarian families, whereas the school performance of European youths who belonged to authoritative parents was better than European youths of authoritarian parents. And she suggested the researchers, practitioners and educators not to treat authoritative parenting as a prototype for some Asian American groups. Chao and Tseng (2002) contended that the merit of this parenting style could reflect only Western groups, excluding Asian groups. However, the point of paradox for consequences of authoritative parenting is usually situated in the area of academic performance. Steinberg, Dornbusch and Brown (1992) proved that adolescents of authoritative parents had better psychosocial development which reflected self-reliance, work orientation, self- esteem, and personal and social competence; less psychological distress including anxiety, depression, and psychosomatic complaints; and less behaviour problems including drug and alcohol use, delinquency, susceptibility to antisocial peer pressure, and school misconduct, in all ethnic groupsWhite-American, Asian- American, African-American and Hispanic-American. 1. Introduction P i h l , p Questioning the benefit of authoritative parenting in a non-Western setting, the present study intended to investigate, in Myanmar, the relationship between parenting styles and strengths of humanity which was a core virtue of character strengths and not related to school performance. Character strengths are a family of morally positively valued traits which are reflected in thoughts, feelings and behaviours, and related to several positive outcomes (Park, 2004; Ruch, Martínez-Martí, Proyer, & Harzer, 2014). In their handbook for character strengths published in 2004, Peterson and Seligman introduced 24 strengths grouped under six universal virtues: (1) wisdom and knowledge, (2) courage, (3) humanity, (4) justice, (5) temperance, and (6) transcendence. In page 29 and 30 of the handbook, Peterson and Seligman (2004) gave explanation for the meaning and composition of the six virtues. Wisdom and knowledge characterize cognitive strengths which give rise to knowledge pursuit and applying knowledge into human affairs, consisting of five strengths: creativity, curiosity, critical thinking, love of learning and perspective. Courage represents emotional strengths which comprise the exercise of will for accomplishing goals against external or internal opposition, and includes four strengths: bravery, persistence, integrity and zest. For the virtue of humanity, the composition of three strengthslove, kindness and social intelligencereflects interpersonal strengths of caring others and fostering familiarity. As virtue of justice, three strengthsteamwork, fairness and leadershipportray civic strengths of favouring equity within society. The virtue of temperance is comprised of four strengthsforgiveness, modesty, prudence and self-regulationwhich facilitate controlling over excess. The last virtue, transcendence, consists of appreciation of beauty and excellence, gratitude, hope, humour and religiousness, representing the strengths which provide people with meaning of living through the connections to the larger universe (or something higher such as God). Research on character strengths has already proved that 24 character strengths had a number of important links to positive outcomes (e.g., academic achievement, life satisfaction, and well-being) in children and youth (Park, 2009; Niemiec, 2013). In the study of Wagner and Ruch (2015), nine character strengths including social intelligence showed positive correlations with classroom behaviour and school achievement. High social intelligence as a strength of humanity lets an individual aware of not only the mind of others but also himself. This benefits people to possess smoother social functioning (Peterson & Seligman, 2004). Another strength of humanity, kindness, is what we all expect from everyone in the world. 1. Introduction P i h l Parenting have long been studied and discussed in social psychological field, and many scholars (e.g., Champney, 1941; Schaefer, 1959; Baumrind, 1968; Rohner, 1976; Buri, 1991; Sessa, Avenevoli, Steinberg, & Morris, 2001) introduced different aspects of parenting which were labelled as parenting practices or factor or dimension or styles. Based on the previous findings and discussions, many theoretical frameworks have been developed and refined to grab the holistic nature of parenting. For example, in the article “The measurement of parent behaviour”, Champney (1941) introduced the three parenting behavioursdemocracy in the home, acceptance of child and indulgence. Then, Baldwin (1955) identified nine aspects of parenting behaviourswarmth, possessiveness, democracy, intellectuality, restrictiveness, severity, interference, adjustment and activeness. Trying to conceptualize parenting based on the parenting behaviour of democracy and acceptance, many researchers proposed new frameworks (e.g., Lorr & Jenkins, 1953; Slater, 1962; Roe & Siegelman, 1963). In 1968, Baumrind proposed three parenting styles: Authoritarian, Authoritative and Permissive. Following her work, Maccoby and Martin (1983) suggested an additional parenting style “Negligent” and these four parenting styles became popular in parenting studies. They posited that two dimensions of parentingresponsiveness and demandingness (control)decide these four parenting styles. Authoritative parenting style is composed of high responsiveness and high control; authoritarian parenting style, with low responsiveness and high control; permissive parenting style, with high responsiveness and low control; and indulgent parenting, with low responsiveness and low control. According to Baumrind (1991), the best way of parenting children is balanced nurturance (responsiveness) and limit-setting (control) which can be found in authoritative parenting styles. g ( ) p g y As this classification of parenting style was exact and systematic, many studies used this framework and proved that authoritative parenting was the best parenting style delivering positive outcomes to children. In reviewing the consequences of parenting styles found by Baumrind (1991), Weiss and Schwarz (1996), and Miller, Cowan, Cowan and Hetherington (1993), Darling (1999) concluded that children and adolescents of authoritative parents were more socially and instrumentally competent than their counterparts of non-authoritative parents, together with low behaviour problems. 2. Methodology A survey was conducted to 378 adolescents in three secondary schools under the permission of respective principals. 134 males comprised 35.4% and 244 females comprised 64.6 % of the sample population. The age of participants ranged from 12 to 17 and the mean age was 13.76. Two questionnaires: Scale of Parenting Styles (see Appendix) and 96-item Value in Action Inventory were used to measure parenting styles and the strengths of humanity. Scale of Parenting Styles (SOPS) was developed by Gafoor and Kurukkan (2014), recommending the high relevance of scales for south Asian adolescents. The scale measures adolescents’ perception of parental responsiveness and parental control. 19 items1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36represents parental responsiveness; 19 items3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 38represents parental control. The rating format applied is 5-point Likert Scale: 1 = very wrong, 2 = mostly wrong, 3 = sometimes right, sometimes wrong, 4 = mostly right and 5 = very right. Gafoor and Kurukkan (2014) instructed to categorize high or low parental responsiveness and control using median-split method first and then to identify parenting styles according to high or low level of the two dimensions. 96-items Value in Action Inventory (VIA Youth-96) was developed by Park and Peterson (2006). The inventory consists of 96 items (4 items per each strength), measuring 24 character strengths through 5-point Likert Scale: 1 = not like me at all, 2 = a little like me, 3 = somewhat like me, 4 = mostly like me and 5 = very much like me. The data of all participants were sent to VIA institute and the aggregate scores for individual strengths were requested. In the present study, the data of only humanity strength were utilized. One week before questionnaire administration, consent forms which included the explanation of research purpose and confidentiality issues were handed out to participants in class. They were requested to discuss with parents for participation and informed that they could choose non-participation for any sort of reasons. On the day of questionnaire administration, the participants were recruited class by class and gathered in a vacant classroom of respective school. After explaining questionnaire rating formats, the questionnaire completion process was started. The whole process took approximately 45 minutes. 1. Introduction P i h l Kindness, also viewed as altruism, is the primary motive behind volunteerism which benefits every society (Peterson & Seligman, 2004). The last strength “love”, representing the capacity to love and be loved, helps a person in forging secure attachment, trust and intimacy (Peterson & Seligman, 2004). Typically, these three strengths “love, kindness and social intelligence” are highly related to the social aspect of life, bringing positive relations with others and making a person more humane, as they were grouped under the virtue of humanity. In the development of good characters which bring in the positive life outcomes to everyone, many factors make contribution. For example, good parenting, family and peer intimacy, positive role models, positive institutions, and youth development programs have important impact on the development of character strengths (Park, 2004). Investigating the effect of parental boding on eight character strengths of adolescents in Hong Kong, Ngai (2015) found the positive effect of parental care and the negative effect of parental control on the three strengths of humanity, integrity, bravery, persistence, fairness and self-regulation. According to Darling (1999), parental care (responsiveness) is related with social competence and higher psychological functioning, while parental control (demandingness) predicts instrumental competenceacademic performanceand behavioural 28 Research on Humanities and Social Sciences www.iiste.org controlless involvement in problematic behaviour. Although the parental responsiveness result of Ngai (2015) was in line with the proposition of Darling (1999), contradiction was found in the negative effect of parental control on character strengths. Moreover, Ngai (2015) did not investigate the combined effect of parental care and parental control, i.e., as parenting styles. p g y The present study replicated the work of Ngai (2015) with a focus on strengths of humanity and in different setting, proposing the following hypotheses: g p p g g yp 1) Parental responsiveness will have significant positive effect on strengths of humanity. 2) Parental control will have significant negative effect on strengths of humanity. (2) Parental control will have significant negative effect on strengths of humanity. 3) The strengths of humanity will be higher in the children of authoritative parents than those of no authoritative parents. (3) The strengths of humanity will be higher in the children of authoritative parents than those of non- authoritative parents. 2. Methodology The gathered data were coded into SPSS data file. Before hypothesis testing, exploratory factor analysis and analysis of internal consistency were executed to SOPS, so as to maximize the effectiveness of SOPS in Myanmar context. Hypotheses were answered by applying SPANOVA, standard multiple regression and MANOVA in data analysis. 3. Data Analysis and Results 7 items from control dimensions (5, 7, 9, 11, 13, 17 and 29) were also removed to balance the number of items in two dimensions. Although any 7 items could be removed from control dimension, those items were selected according to the relatedness in the meaning of 7 responsiveness items. 24 items, i.e., 12 for responsiveness and 12 for control, were remained. The internal consistency was 0.759 for maternal responsiveness, 0.768 for maternal control, 0.746 for paternal responsiveness and 0.773 for paternal control; and the maximum alpha if item deletion was executed was 0.751, 0.768, 0.743 and 0.767 respectively. Item deletion did not produce higher alpha than that of original 12 items. Hence, data of 24 items were used for hypothesis testing. Table 1. Rotated Component Matrix of Maternal Parenting Items Component 1 Component 2 Items Component 1 Component 2 M-17 0.592 M-33 0.395 M-31 0.581 M-10 0.369 0.322 M-29 0.559 M-3 0.368 M-23 0.543 M-15 0.362 M-25 0.541 M-21 0.359 M-35 0.535 M-7 M-9 0.529 M-1 0.695 M-32 0.517 0.333 M-4 0.583 M-38 0.513 M-16 0.578 M-34 0.51 M-12 0.558 M-19 0.51 M-14 0.512 M-13 0.507 M-8 0.314 0.478 M-37 0.506 M-2 0.456 M-30 0.503 M-18 0.451 M-11 0.476 M-36 0.443 M-22 0.457 M-20 0.414 M-5 0.457 M-6 0.41 M-27 0.448 M-24 0.384 M-26 0.411 0.319 M-28 0.335 0.346 First, parenting differences according to parent-child gender were examined through SPANOVA (split-plot ANOVA). The four parenting featuresmaternal responsiveness, maternal control, paternal responsiveness and paternal controlwere treated as within-subject factors, and gender of children was treated as between-subject factor. According to Pallant (2011), sample size of above 30 enables the analysis of variance techniques to be tolerant of violation of normality assumption and the present study possessed a sample size of 378. Box’s M test was significant F (10, 355917.074) = 2.356, p = 0.009, indicating the violation of the assumption for equal covariance across groups. According to Tabachnick and Fidell (2007), Pillai’s Trace was more robust in the presence of assumption violations, and hence, Pillai’s Trace statistic was used for assessment of significant effect. The statistic showed significant main effect of parenting F (3, 374) = 33.842, p < 0.001, while there was no interaction between parenting and child-gender F (3, 374) = 1.725, p = 0.161. 3. Data Analysis and Results y First, exploratory factor analysis (EFA) was conducted for the relevant usage of SOPS in Myanmar context. Running EFA demands two requirements for suitability of the data: adequate sample size and the strength of inter- item relationship (Pallant, 2011). Nunnally (1978) recommended the minimum value of participants-to-item ratio as 10 for running factor analysis appropriately and the ratio was 9.95 (378/ 38) in the present study. Barlett’s test of sphericity was significant (p < 0.001). Kaiser-Meyer-Olkin (KMO) measure of sampling adequacy produced a value of 0.879 which was greater than 0.8a meritorious sampling adequacy according to Hutcheson and Sofroniou (1999). Hence, the results proved satisfactory sample size for applying EFA. Secondly, the inter-item correlation of SOPS was examined. According to Field (2013), the variables which had lots of correlations below 0.3 in correlation matrix should be removed. However, he warned about the term he used“about 0.3” and “lots of”which was not definite and clear, suggesting the readers to act according to circumstances. In the present study, the criterion of removing item was set at no correlations of above 0.25, in order to avoid being too stringent. Every item was correlated with at least another one item above 0.25 and item deletion was not necessary. y y Principal component analysis (PCA) was applied because PCA is a better method to explore “empirical summary of data set” according to Tabachnick and Fidell (2007, p. 635). Varimax rotation technique was selected because varimax is a technique of orthogonal rotation approach preferred for uncorrelated factor solutions (Field, 2013) and the two dimensions responsiveness and controlwere assumed as independent to each other. Items were extracted into two components (see Table 1 and 2). In maternal parenting, besides cross loadings (item 10, 29 Research on Humanities and Social Sciences www.iiste.org iiste.org ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.8, 2019 26 and 32 loaded on both factors), 6 items of the responsiveness dimension10, 22, 28, 30, 32 and 34were loaded on control dimension. According to the rule of Tabachnick and Fidell (2007), a cross loaded item has loading value of 0.32 or higher for two or more factors. In paternal parenting, there was no cross loading but 6 items of the responsiveness dimension22, 26, 28, 30, 32 and 34were loaded on control dimension. To maintain a clear factor structure, those 7 items10, 22, 26, 28, 30, 32 and 34were removed. 3. Data Analysis and Results The comparison of main effect showed slightly higher maternal responsiveness and control than that of father: according to the criteria of Cohen (1988), “d” value of below 0.5 reflects small effect size, and the parent-gender effect on responsiveness and control established of 0.137 and 0.237 (see Table 3). Although the responsiveness and control of mother were higher than that of father, parental responsiveness and care were not different for boys and girls. Second, the effect of parenting dimensions on strengths of humanity was investigated through standard multiple regression analysis because standard regression technique is useful in exploring how much variance in a dependent variable can be explained by a group of independent variables, according to Pallant (2011). Required assumptions for multiple regression analysissample size; mulitcollinearity and singularity; outliers; and normality, linearity, homoscedasticity and independence of residualswere examined first as suggested by Pallant (2011). Stevens (1996) suggested the participant-to-predictor ratio as 15 for reliable regression equation and the ratio was 25.2 in the present study. To examine the existence of multicollinearity among the parenting dimensions, bivariate correlation analysis was conducted. According to Field (2013), correlation of above 0.8 was the sign of multicollinearity and the results in Table 4 showed that the maximum amount of correlation among parenting dimensions was 0.772. For deleting the cases with univariate outliers, the humanity strength variables were first 30 Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.8, 2019 www.iiste.org examined, in order for usage of the resulted data in running MANOVAfor the effect of parenting styles on humanity strengths. 6 participants were detected to provide extreme scores, getting removed from the sample, and the data of 372 was maintained. Then, the outliers of parenting dimensions were identified and further removal was done to 5 participants, remaining the data of 367 participants. T bl 2 R d C M i f P l P i Table 2. 3. Data Analysis and Results Rotated Component Matrix of Paternal Parenting Items Component 1 Component 2 Items Component 1 Component 2 F-31 0.633 F-11 0.42 F-17 0.62 F-21 0.405 F-38 0.568 F-19 0.402 F-29 0.558 F-3 0.384 F-35 0.533 F-15 0.32 F-37 0.528 F-7 F-9 0.508 F-1 0.674 F-13 0.508 F-16 0.593 F-32 0.507 F-4 0.581 F-30 0.497 F-14 0.511 F-26 0.474 F-18 0.505 F-23 0.474 F-12 0.485 F-22 0.471 F-24 0.468 F-28 0.466 F-6 0.453 F-27 0.461 F-20 0.422 F-5 0.449 F-8 0.421 F-33 0.445 F-36 0.404 F-25 0.444 F-10 0.343 F-34 0.433 F-2 0.337 Finally, regression was run for initial checks. The Normal P-P Plot produced a straight line and the scatter plot demonstrated rectangularly distributed and centre-concentrated points for all regression analysis, providing evidence for normality and linearity of residuals. Regarding outliers with standardized residual value of above ±3.3, there were no cases in analysis for love, only 1 case in analysis for kindness, and one case, too, in analysis for social intelligence, indicating that the percentage of residual outliers was acceptable: less than 1% of the whole sample. However, five participants had the Mahalanobis distance of above critical value (18.47) and after removing them, the data of 362 participants were kept. The maximum value of Cook’s distance was 0.035, 0.072 and 0.91, respectively for love, kindness and social intelligence: all values were lower than 1, indicating absence of cases that might influence regression model. Using the data of 362 participants, parenting dimensions were regressed on love, kindness and social intelligence separately (see Table 5). Only maternal responsiveness predicted significantly for love and social intelligence, and only paternal control had significant prediction for kindness. Holistically, the parenting dimensions explained 11.3% variance in love, 11.6% variance in kindness and 9% variance in social intelligence of children. g Table 3. Comparison of Parenting Dimensions in Terms of Parent-Gender Parenting Gender Mean Mean Difference p (Bonferroni adjusted) Responsiveness Mother 43.38 1.084 0.006 0.137 Father 42.42 Control Mother 46.01 1.474 < 0.001 0.237 Father 44.44 31 Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.8, 2019 Table 4. Bivariate Correlation of Parenting Dimensions Paternal Responsiveness Maternal Control Paternal Control Maternal Responsiveness .620 .467 .385 Paternal Responsiveness .415 .464 Maternal Control .772 ** Correlation is significant at the 0.01 level (2-tailed). Table 5. 3. Data Analysis and Results The Prediction of Parenting Dimensions on Strengths of Humanity Love Kindness Social Intelligence Constant 1.93 2.576 2.516 Maternal Responsiveness (β) 0.156* 0.073 0.14* Paternal Responsiveness (β) 0.003 0.041 0.033 Maternal Control (β) 0.129 0.12 0.079 Paternal Control (β) 0.12 0.174* 0.118 0.113 0.116 0.09 F (4, 327) 11.36 11.721 8.8 Sig. <0.001 <0.001 <0.001 Third, the effect of parenting styles on strengths of humanity was investigated through MANOVA, counting in the gender effect together with maternal and paternal parenting styles. Four types of parenting styles which included negligent, permissive, authoritarian and authoritative were identified according to the guideline of Gafoor and Kurukkhan (2014). For important assumption checks required for MANOVA, the procedure suggested by Pallant (2011) was followed. According to her, the number of participants in each group32 (4 × 4 × 2) groups in the present studymust be larger than the number of dependent variables. Among 32 groups, some were found to have only 1 participant and also among 16 groupswhen parenting styles of mother and father were pairedone-participant groups still appeared. Only when gender was paired with maternal parenting styles or paternal parenting styles, the minimum number of participants in groups was 55 and assumption for sample adequacy was met. Consequently, the interaction between parenting styles of mother and father was not taken into account. MANOVA was run separately for mother and father. Using Bonferroni correction, the alpha level was set at 0.025. Third, the effect of parenting styles on strengths of humanity was investigated through MANOVA, counting in the gender effect together with maternal and paternal parenting styles. Four types of parenting styles which included negligent, permissive, authoritarian and authoritative were identified according to the guideline of Gafoor and Kurukkhan (2014). For important assumption checks required for MANOVA, the procedure suggested by Pallant (2011) was followed. According to her, the number of participants in each group32 (4 × 4 × 2) groups in the present studymust be larger than the number of dependent variables. Among 32 groups, some were found to have only 1 participant and also among 16 groupswhen parenting styles of mother and father were pairedone-participant groups still appeared. Only when gender was paired with maternal parenting styles or paternal parenting styles, the minimum number of participants in groups was 55 and assumption for sample adequacy was met. Consequently, the interaction between parenting styles of mother and father was not taken into account. 3. Data Analysis and Results η Parenting Styles of Mother Love 10.227 3 364 <0.001 .078 Kindness 9.787 3 364 <0.001 .075 Social Intelligence 9.873 3 364 <0.001 .075 Parenting Styles of Father Love 7.305 3 364 <0.001 .053 Kindness 6.770 3 364 <0.001 .075 Social Intelligence 7.801 3 364 <0.001 .071 Th i lt b d B f i ti ( T bl 9 d 10) l d th ti Table 7. The Effect of Parenting Styles and Gender on Strength of Humanity Effect Pillai’s Trace F Hypothesis df Error df Sig. Maternal Parenting Styles .158 6.757 9.000 1092.000 <0.001 .053 Paternal Parenting Styles .860 6.254 9.000 881.163 <0.001 .049 Gender .017 2.106 3.000 362.000 .099 .017 Maternal Parenting Styles * Gender .022 .913 9.000 1092.000 .513 .007 Paternal Parenting Styles * Gender .972 1.143 9.000 881.163 .329 .009 According to Table 8, parenting styles of both mother and father had moderate significant effect on the three ording to Table 8, parenting styles of both mother and father had moderate significant effect on the three of humanity, except the low moderate effect of father’s parenting style on love. Th Eff f P i S l L Ki d d S i l I lli According to Table 8, parenting styles of both mother and father had moderate significant effect on the three strengths of humanity, except the low moderate effect of father’s parenting style on love. Table 8. The Effect of Parenting Styles on Love, Kindness and Social Intelligence Independent Variable Dependent Variable F Hypothesis df Error df Sig. η Parenting Styles of Mother Love 10.227 3 364 <0.001 .078 Kindness 9.787 3 364 <0.001 .075 Social Intelligence 9.873 3 364 <0.001 .075 Parenting Styles of Father Love 7.305 3 364 <0.001 .053 Kindness 6.770 3 364 <0.001 .075 Social Intelligence 7.801 3 364 <0.001 .071 The group comparison results based on Bonferroni correction (see Table 9 and 10) revealed the negative effect of negligent parenting. In all three strengths of humanity, the children of negligent parenting established significantly lower scores than the rest parenting styles. The level of love in children of negligent mothers was lower than those of permissive and authoritative mothers. Similarly, the feeling of love in children of negligent fathers was lower than those of authoritarian and authoritative fathers. In the strength of kindness, children of authoritative parents were higher than those of negligent and permissive parents. 4. Discussion and Conclusion The main purpose of this study was to explore whether the merit of authoritative parenting styles could be found in Myanmar, an Asian society, using strengths of humanity as indicator which would reflect the merit. Specifically, the present study investigated the effect of parent-child gender type on parenting; the effects of parenting 3. Data Analysis and Results MANOVA was run separately for mother and father. Using Bonferroni correction, the alpha level was set at 0.025. As univariate outlier case was already solved in multiple regression analysis, multivariate normality was examined through Mahalanobis distance. Only one participant has the value of Mahalanobis distance (21.66) greater than critical value (16.27). Expecting the tiny effect of one participant, removing was abstained. The Pearson correlation coefficients of three humanity strength variables (see Table 6) proved the absence of multicolinearity. y Table 6. Bivariate Correlation of Humanity Strength Variables Kindness Social Intelligence Love .225 .188 Kindness .262 Correlation is significant at the 0.01 level (2-tailed). g ( ) In MANOVA for father’s parenting style effect, the test of Box’s M testified to the equal covariance matrices across groups F (42, 62567.159) = 1.345, p = 0.067, while the test was significant in MANOVA for mother’s parenting style effect F (42, 58165.403) = 1.680, p = 0.004, and hence, Pillai’s Trace statistic was used in assessment of significant effect. The statistic showed significant main effect of parenting styles of both mother and father with small effect size: according to the criteria of Cohen (1988), “ ” value of 0.02 to 0.06 reflects small effect size, and value above 0.06 reflects moderate effect size (see Table 7). Gender effect did not exist for humanity strength variables, both in terms of main and interaction effect. 32 ww.iiste.org Table 7. The Effect of Parenting Styles and Gender on Strength of Humanity Effect Pillai’s Trace F Hypothesis df Error df Sig. Maternal Parenting Styles .158 6.757 9.000 1092.000 <0.001 .053 Paternal Parenting Styles .860 6.254 9.000 881.163 <0.001 .049 Gender .017 2.106 3.000 362.000 .099 .017 Maternal Parenting Styles * Gender .022 .913 9.000 1092.000 .513 .007 Paternal Parenting Styles * Gender .972 1.143 9.000 881.163 .329 .009 According to Table 8, parenting styles of both mother and father had moderate significant effect on the three strengths of humanity, except the low moderate effect of father’s parenting style on love. Table 8. The Effect of Parenting Styles on Love, Kindness and Social Intelligence Independent Variable Dependent Variable F Hypothesis df Error df Sig. 4. Discussion and Conclusion sion and Conclusion purpose of this study was to explore whether the merit of authoritative parenting styles could be found 3. Data Analysis and Results Specifically, the present study investigated the effect of parent-child gender type on parenting; the effects of parenting 33 Research on Humanities and Social Sciences Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.8, 2019 www.iiste.org dimensions (parental responsiveness and parental control) on the strengths of humanity; and the effect of parenting styles on the strengths of humanity. dimensions (parental responsiveness and parental control) on the strengths of humanity; and the effect of parenting styles on the strengths of humanity. First, the parenting differences in terms of parent-child gender were explored and it was found that Myanmar adolescents perceived their mothers as more responsive and more controlling to them than their fathers. The result supported the finding of Shek (2000): fathers were perceived as less responsive and less demanding, and the result was also indirectly in line with the proposition of Patterson (1982) about mother’s more engagement in disciplinary actions in that disciplining also includes control and demands. However, perception with parental responsiveness and control was not different between boys and girls. The result was contradicted with the findings of many studies which proved more control of fathers on son (Xie, 1998; Trommsdorff and Iwawaki, 1989; Rohner & Pettengill, 1985) and with the claims of Leaper (2002) about more maternal responsiveness to girls. Then, to answer Hypothesis No (1) and (2), the maternal responsiveness, maternal control, paternal responsiveness and paternal control were regressed on strengths of humanity. Only maternal responsiveness predicted love and kindness, while social intelligence was positively predicted only by paternal control. Regardless of significance, all parenting dimensions showed positive relations with strengths of humanity. Hence, the proposition of Hypothesis No.2 was nullified. Hypothesis No.1 cannot be fully verified, too. The positive effect of maternal responsiveness on love and kindness strengths of adolescents supported the finding of Ngai (2015): parental care positively predicted integrity, bravery, persistence, kindness, love, social intelligence, fairness and self-regulation. On the contrary, the positive effect of paternal control on social intelligence of adolescents was against the results that Ngai (2015) obtainedthe negative effect of parental control on eight character strengths. The present study found that both parenting dimensions were positively related to strengths of humanity. p y p g p y g y Finally, the effect of four parenting styles (negligent, authoritarian, permissive and authoritative) on strengths of humanity was investigated and the results proved Hypothesis No.3. 3. Data Analysis and Results In social intelligence, children of negligent mothers were lower than those of authoritative mother, while children of negligent fathers were lower than those of fathers who practiced the other parenting stylesauthoritative, authoritarian and permissive. than those of fathers who practiced the other parenting styles authoritative, authoritarian and permissive. Table 9. Bonferoni Adjusted Pairwise Comparison for the Effect of Maternal Parenting Styles Dependent Variable Parenting Style of Mother Mean Difference (I-J) Std. Error Sig (I) (J) Love Score Negligent Permissive -.274* 0.099 0.037 Negligent Authoritative -.428* 0.078 <0.001 Kindness Negligent Authoritative -.339* 0.063 <0.001 Permissive Authoritative -.234* 0.082 0.028 Social Intelligence Negligent Authoritative -.378* 0.07 <0.001 Table 10. Bonferoni Adjusted Pairwise Comparison for the Effect of Paternal Parenting Styles Dependent Variable Parenting Style of Father Mean Difference (I-J) Std. Error Sig (I) (J) Love Score Negligent Authoritarian -.264* 0.097 0.042 Negligent Authoritative -.344* 0.079 <0.001 Kindness Negligent Authoritative -.332* 0.063 <0.001 Permissive Authoritative -.249* 0.08 0.012 Social Intelligence Negligent Authoritarian -.250* 0.086 0.022 Negligent Permissive -.241* 0.087 0.034 Negligent Authoritative -.357* 0.07 <0.001 4 Di i d C l i p p g y , p Table 9. Bonferoni Adjusted Pairwise Comparison for the Effect of Maternal Parenting Styles Dependent Variable Parenting Style of Mother Mean Difference (I-J) Std. Error Sig (I) (J) Love Score Negligent Permissive -.274* 0.099 0.037 Negligent Authoritative -.428* 0.078 <0.001 Kindness Negligent Authoritative -.339* 0.063 <0.001 Permissive Authoritative -.234* 0.082 0.028 Social Intelligence Negligent Authoritative -.378* 0.07 <0.001 Table 10. Bonferoni Adjusted Pairwise Comparison for the Effect of Paternal Parenting Styles Dependent Variable Parenting Style of Father Mean Difference (I-J) Std. Error Sig (I) (J) Love Score Negligent Authoritarian -.264* 0.097 0.042 Negligent Authoritative -.344* 0.079 <0.001 Kindness Negligent Authoritative -.332* 0.063 <0.001 Permissive Authoritative -.249* 0.08 0.012 Social Intelligence Negligent Authoritarian -.250* 0.086 0.022 Negligent Permissive -.241* 0.087 0.034 Negligent Authoritative -.357* 0.07 <0.001 4. Discussion and Conclusion The main purpose of this study was to explore whether the merit of authoritative parenting styles could be found in Myanmar, an Asian society, using strengths of humanity as indicator which would reflect the merit. 5. Limitation and Recommendation for Future Research 5. Limitation and Recommendation for Future Research The first limitation of this study was convenient sampling. Participants were recruited from secondary schools for the sake of convenience, and the generalizability of findings would exclude Myanmar adolescents who were not students. The second limitation was application of only one type of reporters: the adolescents. Leung, Daniel and Shek (2014) revealed the negative effect of “the discrepancies in perception of parents and adolescents with parenting characteristics” on developmental outcomes of adolescents. Measuring parenting practices from the perception of both parents and children would help the future researchers to pursue more extensive understanding on the relations of parenting and outcome variables. The third limitation was cross-sectional design of the study. The onset of parenting process comes right after the birth of a person and parenting characteristic changes across the developmental stage of the offspring. Longitudinal studies could provide more comprehensive findings with the effect of parenting. Baldwin, A. L. (1955). Behavior and development in childhood. New York: Dryden Press. 3. Data Analysis and Results Adolescents of authoritative mothers possessed higher strengths of humanity than those of negligent mothers; and higher kindness than those of permissive mothers. Similar result was found with authoritative fathers. Adolescents of authoritative fathers showed higher strengths of humanity than those of negligent fathers; and higher kindness than those of permissive fathers. Adolescents of negligent mothers also had lower level of love than those of permissive mothers, while adolescents of negligent fathers revealed lower level of love than those of authoritarian fathers. In social intelligence, adolescents of negligent father were lower than their counterparts of parents who practiced authoritative, authoritarian and permissive styles. All in all, authoritative parenting style was better than the rest styles; negligent style was the worst; while permissive and authoritarian styles did not show significant differences for the strengths of humanity. The results were in line with what Darling (1999) claimed about the consequences of parenting styles: children and adolescents of authoritative parents performed better than non-authoritative parents in the domains of social competence, academic achievement, psychosocial development, and involvement in problematic behaviour, while those of negligent parents showed poorest performance in all domains. The results also proved the benefit of authoritative parenting styles in an Asian society. On the contrary of the gender difference in character strengths found by Park and Peterson (2006) and Shimai, Otake, Park, Peterson and Seligman (2006), i.e., females tended to report strengths of love and kindness more than males, no gender effect on strengths of humanity was found in the present study. Gender produced no interaction effect with parenting and also it was already described above that gender effect did not exist for adolescents’ perception with parental responsiveness and control. The reason of non-significant gender effect might be the big gap between the number of male and female participants. Male comprised only 35.4% of the sample. To be concluded, the present study proved that both of parental responsiveness and parental control, as well as their combined effect (parenting styles of both parents) played an important role in the development of strengths of humanity. References References Baldwin, A. L. (1955). Behavior and development in childhood. New York: Dryden Press. 34 Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.8, 2019 Research on Humanities and Social Sciences www.iiste.org Baumrind, D. (1968). Authoritarian vs. authoritative parental control. Adolescence, 3, 255–272. Baumrind, D. (1991). The influence of parenting style on adolescent competence and substance use. J Early Adolescence, 11(1), 56-95. Buri, J. R. (1991). Parental Authority Questionnaire. Journal of Personality Assessment, 57, 110–119. Champney, H. (1941). The measurement of parent behavior. Child Development, 12, 131 166. Chao, R. (2001). Extending research on the consequences of parenting style for Chinese Americans and European Americans. Child Development, 72, 1832-1843. Chao, R., & Tseng, V. (2002). Parenting of Asians. In M. H. Bornstein (Series ed.), Handbook of parenting: Vol. 4, Social conditions and applied parenting (2nd ed., pp. 59-93). Mahwah, New Jersey: Lawrence Erlbaum Associates. Cohen, J. W. (1988). Statistical power analysis for the behavioral sciences (2nd ed). Hillsdale, NJ: Lawrence Erlbaum Associates. Darling, N. (1999). Parenting style and its correlates. ERIC Digest, 1-7. Dornbusch, S. M., Ritter, P. L., Leiderman, H. P., Roberts, D. F, & Fraleigh, M. J. (1987). The relation of parenting style to adolescent school performance. Child Development, 58, 1244-1257. Field, A. (2013). Discovering statistics using IBM SPSS statistics (4th ed). London: Sage publications. Gafoor, K. A., & Kurukkan, A. (2014). Construction and validation of scale of parenting style. Guru J Behavioral and Social Sciences, 2(4), 315-323. ( ) Hutcheson, G., & Sofroniou, N. (1999). The multivariate social scientist. London: Sage. Leaper, C. (2002). Parenting girls and boys. In M. H. Bornstein (Series ed.), Handbook of parenting: Vol. 1, Children and Parenting (2nd ed., pp. 189-225). Mahwah, New Jersey: Lawrence Erlbaum Associates. eape , C. ( 00 ). a e t g g s a d boys. . . o ste (Se es ed.), andbook of pa enting: Vol. , Children and Parenting (2nd ed., pp. 189-225). Mahwah, New Jersey: Lawrence Erlbaum Associates. g ( pp ) y Leung, K., Lau, S., & Lam, W. L. (1998). Parenting styles and achievement: A cross-cultural study. Merrill- Palmer Quarterly, 44, 157-172. Leung, J. T. Y., & Daniel T. L. Shek, D. T. L. (2014). Parent–adolescent discrepancies in perceived parenting characteristics and adolescent developmental outcomes in poor Chinese families. J Child Fam Stud, 23, 200– 213. DOI 10.1007/s10826-013-9775-5 Lorr, M. References 35 Research on Humanities and Social Sciences ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.8, 2019 Research on Humanities and Social Sciences www.iiste.org www.iiste.org Vol.9, No.8, 2019 Rohner, R. P., & Pettengill, S. M. (1985). Perceived parental acceptance–rejection and parental control among Korean adolescents. Child Development, 56, 524–528. Ruch, W., Martínez-Martí, M. L., Proyer, R. T., & Harzer, C. (2014). The Character Strengths Rating Form (CSRF): Development and initial assessment of a 24-item rating scale to assess character strengths. Personality and Individual Differences, 68, 53–58. Schaefer, E. S. (1959). A circumplex model for maternal behavior. Journal of Abnormal and Social Psychology, 59, 226–235. Sessa, F. M., Avenevoli, S., Steinberg, L., & Morris, A. S. (2001). Correspondence among informants on parenting: Preschool children, mothers, and observers. Journal of Family Psychology, 15, 53–68. Shek, D. (2000). Differences between fathers and mothers in the treatment of, and relationship with, their teenage children: Perception of Chinese adolescents. Adolescence, 35, 135-146. Shek, D. (2000). Differences between fathers and mothers in the treatment of children: Perception of Chinese adolescents. Adolescence, 35, 135-146. Shimai, S., K. Otake, K., Park, N., Peterson, C., & Seligman, M. E. P. (2006). Convergence of character in american and japanese young adults. Journal of Happiness Studies, 7(3), 311-322. j p y g f pp ( ) Slater, P. E. (1962). Parental behavior and the personality of the child. Journal of Genetic Psychology, 101, 53– 68. Steinberg, L., Dornbusch, S.M., & Brown, B.B. (1992). Ethnic differences in adolescent achievement: An ecological perspective. American Psychologist, 47, 723-729. d ecological perspective. American Psychologist, 47, 723 729. ns, J. (1996). Applied multivariate statistics for the social sciences (3rd ed). Mahwah, NJ: Lawrence Erlbaum Stevens, J. (1996). Applied multivariate statistics for the social sciences (3rd ed). Mahwah, NJ: Lawrence Tabachnick, B.G., & Fidell, L.S. (2007). Using Multivariate Statistics (5 ed.). New York: Allyn and Bacon. Trommsdorff, G., and Iwawaki, S. (1989). Students’ perceptions of socialization and gender role in Japan and Germany. International Journal of Behavioral Development, 12, 485–493. Wagner, L., & Ruch, W. (2015). Good character at school: Positive classroom behavior mediates the link between character strengths and school achievement. Frontiers in Psychology, 6(24). DOI: 10.3389/fpsyg.2015.00610 Weiss L H & Schwarz J C (1996) The relationship between parenting types and older adolescents' personality Wagner, L., & Ruch, W. (2015). References Good character at school: Positive classroom behavior mediates the link between character strengths and school achievement. Frontiers in Psychology, 6(24). DOI: 10.3389/fpsyg.2015.00610 Weiss, L. H., & Schwarz, J. C. (1996). The relationship between parenting types and older adolescents' personality, academic achievement, adjustment, and substance use. Child Development, 67(5), 2101-2114. Weiss, L. H., & Schwarz, J. C. (1996). The relationship between parenting types and older adolescents' pe academic achievement, adjustment, and substance use. Child Development, 67(5), 2101-2114. Xie, Q. (1998). Perceptions of childrearing practices by Chinese parents and their only children, and their relations to children’s school achievement. Dissertation Abstracts International, 58, 3425. References G., & Jenkins, R. L. (1953). Three factors in parent behavior. Journal of Consulting Psychology, 17, 306– 308. Maccoby, E. E., & Martin, J. A. (1983). Socialization in the context of the family: Parent–child interaction. In P. Mussen, & E. M. Hetherington (Eds.), Handbook of Child Psychology: Socialization, personality, and social development (Vol. 4). New York: Wiley. p ( ) y McBride-Chang, C., & Chang, L. (1998). Adolescent-parent relations in Hong Kong: Parenting styles, emotional autonomy, and school achievement. Journal of Genetic Psychology, 159, 421-436. Miller, N. B., Cowan, P. A., Cowan, C. P., & Hetherington, E. M. (1993). Externalizing in preschoolers adolescents: A cross-study replication of a family model. Developmental Psychology, 29(1), 3-18. Moua, S. (2010). Parenting Styles of Hmong Parents and its Effects and Contributions to Hmong Student's Academic Achievement. A Research Paper Submitted in Partial Fulfillment of the Requirements for the Master of Science Degree, School Counseling, University of Wisconsin-Stout. Ngai, S. S. (2015). Parental bonding and character strengths among Chinese adolescents in Hong Kong. International Journal of Adolescence and Youth, 20(3), 317-333, DOI: 10.1080/02673843.2015.1007879 Niemiec, R. M. (2013). VIA character strengths: Research and practice (The first 10 years). In H. H. Knoop & A. Delle Fave (Eds.), Well-being and cultures: Perspectives on positive psychology (pp. 11-30). New York: Springer. p g Nunnally, J. O. (1978). Psychometric theory. New York: McGraw-Hill. Pallant, J. (2011). SPSS survival manual: A step by step guide to data analysis using the SPSS Program (4th ed). Allen & Unwin: Berkshire. Park, N. (2004). Character strengths and positive youth development. The Annals of the American Academy of Political and Social Science, 591, 40–54. Park, N. (2009). Building strengths of character: Keys to positive youth development. Reclaiming Children and Youth, 18(2) 42-47. Park, N., & Peterson, C. (2006). Moral competence and character strengths among adolescents: The development and validation of the Values in Action Inventory of Strengths for Youth. Journal of Adolescence, 29, 891- 905. Patterson, G. R. (1982). Coercive family process. Eugene, OR: Castlia Press. Peterson, C., & Seligman, M. E. P. (2004). Character strengths and virtues: A handbook and classification. New York, NY: Oxford University Press. Roe, A., & Siegelman, M. (1963). A parent-child relations questionnaire. Child Development, 34(2), 355–369. Rohner, R. P. (1976). They love me, they love me not: A worldwide study of the effects of parental acceptance and rejection. New Haven, CT: Yale University Press. Appendix 1. My mother/father does whatever I tell. 1. My mother/father does whatever I tell. 2. My mother/father spends free time with me. 3. My mother/father points out my mistakes in the manner that I understand. 4. My mother/father gives money for my needs. 4. My mother/father gives money for my needs. 5. My mother/father discusses the benefits and detriments of my learning topics. 5. My mother/father discusses the benefits and detriments of my learning topics. 5. My mother/father discusses the benefits and d 6. My mother/father considers my likes in food. 7. My mother/father controls my game when in excess. . My mother/father enquires the reason for my failure. 10. My mother/father helps me in studying. y p y g 11. My mother/father confers responsibilities in accordance with my growth. 13. My mother/father enquires the reasons for reaching home late 14. My mother/father accepts my privacy 15. My mother/father takes care of my dressing 16. My mother/father fulfils my desires with available means. 17. My mother/father makes me aware that the responsibility of what i do is mine itself. 18. My mother/father accepts when i say no to what i dislike. 19. My mother/father tells how i should behave with their friends. My mother/father talks to me praising about their frien 21. My mother/father tries to frame my likes and dislikes. 22. My mother/father appreciates when i try to become independent. 22. My mother/father appreciates when i try to be 23. My mother/father punishes for my mistakes. 24. My mother/father shows love when i do any mistake. 5. My mother/father enquires who my friends are. My mother/father has given me freedom to select the subject for study. 27. My mother/father organizes time for my play. 28. My mother/father gives priorities to my preferences in studies. 8. My mother/father gives priorities to my preferenc 29. My mother/father demands me to be systematic in studies. 30. My mother/father emphasizes my successes. 36 ISSN 2224-5766 (Paper) ISSN 2225-0484 (Online) DOI: 10.7176/RHSS Vol.9, No.8, 2019 31. My mother/father advices me. 31. My mother/father advices me. 32. My mother/father celebrates in my successes with me. 33. My mother/father discourages unhealthy foods. 34. My mother/father gets anxious when i am late to reach home. 5. My mother/father inquires how i spend money. 36. My mother/father buy dresses for me according to the latest trends. 37. My mother/father enquires how i spend my free time. 38. Appendix My mother/father gives me timely advices. 37
https://openalex.org/W2989615293	https://www5.bahiana.edu.br/index.php/evidence/article/download/2510/2961	English	null	HPV vaccines for prevention of cervical pre-cancer in adolescent girls and women	Journal of Evidence-Based Healthcare	2,019	cc-by	1,749	How to cite this article: Li JJ, Stetz J. HPV vaccines for prevention of cervical pre-cancer in adolescent girls and women. J Évid-Based Healthc. 2019;1(2):112-115. doi: 10.17267/2675-021Xevidence.v1i2.2510 How to cite this article: Li JJ, Stetz J. HPV vaccines for prevention of cervical pre-cancer in adolescent girls and women. J Évid-Based Healthc. 2019;1(2):112-115. doi: 10.17267/2675-021Xevidence.v1i2.2510 How to cite this article: Li JJ, Stetz J. HPV vaccines for prevention of cervical pre-cancer in adolescent girls and women. J Évid-Based Healthc 2019;1(2):112-115. doi: 10.17267/2675-021Xevidence.v1i2.2510 How to cite this article: Li JJ, Stetz J. HPV vaccines for prevention of cervical pre-cancer in adolescent girls and women. J Évid-Based Healthc. 2019;1(2):112-115. doi: 10.17267/2675-021Xevidence.v1i2.2510 Critical appraisal of evidence HPV vaccines for prevention of cervical pre-cancer in adolescent girls and women HPV vaccines for prevention of cervical pre-cancer in adolescent girls and women Jia Jian Li1, Jessica Stetz2 1Corresponding author. Downstate Medical Center, State University of New York. New York City, New York, United States of America. ORCID: 0000-0002-4446-9412. jiajian.li@downstate.edu 2Downstate Medical Center, State University of New York. New York City, New York, United States of America. ORCID: 0000-0001-6183-2925. stetzjessica@yahoo.com 1Corresponding author. Downstate Medical Center, State University of New York. New York City, New York, United States of America. ORCID: 0000-0002-4446-9412. jiajian.li@downstate.edu 2Downstate Medical Center, State University of New York. New York City, New York, United States of America. ORCID: 0000-0001-6183-2925. stetzjessica@yahoo.com Submitted 09/08/2019, Accepted 10/03/2019, Published 10/21/2019 J. Évid-Based Healthc., Salvador, 2019 December;1(2):112-115 Doi: 10.17267/2675-021Xevidence.v1i2.2510 \| ISSN: 2675-021X Submitted 09/08/2019, Accepted 10/03/2019, Published 10/21/2019 J. Évid-Based Healthc., Salvador, 2019 December;1(2):112-115 Doi: 10.17267/2675-021Xevidence.v1i2.2510 \| ISSN: 2675-021X Submitted 09/08/2019, Accepted 10/03/2019, Published 10/21/2019 J. Évid-Based Healthc., Salvador, 2019 December;1(2):112-115 Doi: 10.17267/2675-021Xevidence.v1i2.2510 \| ISSN: 2675-021X Narrative evaluated as an outcome due to inadequate study sample sizes and insufficient follow up duration2. The summary of the Cochrane review analyses results is presented in the table. Human papillomaviruses (HPV) are sexually transmitted infections that are common in young people. In the United States, HPV infects nearly 80 million people. Approximately, one in four Americans are currently infected with HPV. About 14 million individuals become infected with HPV each year in the United States1. Usually these viruses are cleared by the immune system and pose little threat. However, high- risk (hr) types of HPV can cause persistent infection which can lead to precancerous changes in cells. Precancerous changes are defined as infection of at least two thirds of the surface layer of cervical cells. Some people with this pre-cancerous abnormality will go on to develop cervical cancer; unfortunately, we don’t know which individuals with infection will clear these changes and which will develop cancer. A number of different hrHPV types cause cervical pre-cancer and cancer, the most important of which are HPV16 and 18. These high-risk types cause about 70% of cervical cancers worldwide2. Cervical cancer is the fourth most common cause of cancer in women worldwide, and 86% of worldwide cervical cancer cases occur in developing countries. The lifetime risk of cervical cancer is approximately 0.6% and the chance of dying from cervical cancer is approximately 0.2%3. Vaccination with HPV-like particles triggers the production of antibodies which protect against the development of precancerous cervical changes. Jia Jian Li1, Jessica Stetz2 The Cochrane review discussed here assesses the benefits and harms of prophylactic HPV vaccination against cervical pre-cancer and HPV16/18 infections in adolescent girls and women2. The assumption is that if HPV vaccine effectively prevents precancerous changes by hrHPV infection, vaccination should prevent a majority of cases of cervical cancer. There were no significant differences in serious adverse events between vaccinated and unvaccinated individuals (RR: 0.98, 95% CI 0.92 to 1.05; 71,597 participants; high-quality evidence) independent of type or dosage of HPV vaccine. The HPV vaccine administration was not associated with increased risk of pregnancy-related adverse outcomes either (moderate to high quality evidence). Caveats However, the analyses of bias performed by the Cochrane review found little evidence of important bias due to trial sponsorship. sponsored by the vaccine manufacturers. However, the analyses of bias performed by the Cochrane review found little evidence of important bias due to trial sponsorship. The quality of the evidence was deemed to be high, since the data included more than 70,000 women from randomized trials2. Heterogeneity was minimal in this review since data series did not combine participants with different baseline HPV status (presence of hrHPV DNA, presence of DNA of the HPV vaccine types). Moreover, younger (15 to 26 years) women were distinguished from mid-adult women (24 to 45 years). Efficacy estimates were not significantly different by vaccine type, and jointly pooled estimates were retained. Only when significant heterogeneity by vaccine types was noted, were separate efficacy estimates by vaccine type pooled2. When significant heterogeneity persisted, the review used meta-regression to investigate sources of heterogeneity, such as serological status, study design items, study size and sexual history6,7. This process decreased any heterogeneity presented in the data. While the reason for higher efficacy of HPV vaccine in younger women is not discussed in the Cochrane review, it is likely that younger age allows for more likelihood of exposure to HPV and potentially developing pre-cancerous cervical lesions. This factor could justify the recommendations that HPV vaccines should be administered as early as possible in adolescence. Finally, it is important to remember that the lifetime risk of cervical cancer is approximately 0.6%3. So even if the vaccine is 100% effective at preventing cervical cancer, the best NNT for cancer prevention over a lifetime would be 167. As of December 2018, the cost of HPV vaccine is approximately $261-$461. Caveats Vaccination against HPV is performed to reduce the incidence of cervical cancer. Unfortunately, this outcome could not be assessed in the meta-analysis, because the included trials lacked sufficient power and adequate follow up2. Additionally, this meta- analysis extracted efficacy data from peer-reviewed published reports. Incomplete reporting of the original trials limited the number of studies that could be included in each of the analyses2. Furthermore, HPV vaccines were not yet standardized at the time of the studies so the potency for specific anti-HPV serological responses may be a variable4. The review identified several notable biases. Computed efficacy estimates for women who received only one or two doses were calculated instead of measured. Another post hoc analysis limitation was the variation caused by counting events for participants who received at least one dose at day one, compared to counting events for participants who received all three doses on the day of the last administration2. Another source of bias originated from the fact that for several outcomes no information was available for mid-adult women. Finally, the comparison of the risks of adverse events was compromised by the use of different products administered to participants in the control group, varying from adjuvant (often aluminum hydroxide or other aluminum compound) or an alternative vaccine (often Hepatitis A or Hepatitis B)5, and therefore, the pooled risks of adverse effects associated with HPV vaccines and the assumed risks for control groups must be interpreted carefully. It must be noted that 25 out of the 26 trials were A total of 26 studies involving 73,428 adolescent girls and women were included in the Cochrane meta- analysis. The authors performed pre-planned analyses separately in groups of women 15-25 and 26-45, as the majority of trials enrolled subjects 25 years of age or younger2. Participants were divided into sub-groups; the first group had no evidence of baseline infection with high-risk human papillomaviruses types, the second group had no evidence of baseline infection with HPV types included in the vaccines, and the third group had unknown baseline infection with HPV. The review included all trials that evaluated vaccine safety over a period 0.5 to 7 years and ten trials with follow-up 3.5 to 8 years. Cervical cancer could not be J. Évid-Based Healthc., Salvador, 2019 December;1(2):112-115 Doi: 10.17267/2675-021Xevidence.v1i2.2510 \| ISSN: 2675-021X 113 J. Évid-Based Healthc., Salvador, 2019 December;1(2):112-115 Doi: 10.17267/2675-021Xevidence.v1i2.2510 \| ISSN: 2675-021X 113 sponsored by the vaccine manufacturers. Conclusion The evidence presented in this meta-analysis shows the HPV vaccination confers significant benefit in preventing cervical pre-cancer. The effect is higher for lesions associated with HPV16/18. The data also demonstrates an absence of serious adverse events. Therefore, we have assigned a color recommendation of Green (Benefit > Harm) to this vaccine. 114 J. Évid-Based Healthc., Salvador, 2019 December;1(2):112-115 Doi: 10.17267/2675-021Xevidence.v1i2.2510 \| ISSN: 2675-021X Table. Efficacy of HPV vaccine for preventing cervical pre-cancer in different subgroups based on age and type of cervical abnormality e. Efficacy of HPV vaccine for preventing cervical pre-cancer in different subgroups based on age and type of cervical abnormality Author contributions Li JJ and Stetz J designed the evidence-based review protocol. Li JJ drafted the manuscript. Stetz J reviewed the statistical analysis and edited the manuscript. Competing interests No financial, legal or political competing interests with third parties (government, commercial, private foundation, etc.) were disclosed for any aspect of the submitted work (including but not limited to grants, data monitoring board, study design, manuscript preparation, statistical analysis, etc.). References 1. Centers for Disease Control and Prevention. Papilomavírus Humano (HPV). [Internet]. 2018. [accessed 12 nov. 2018]. Available at: https://www.cdc.gov/hpv/parents/vaccine.html. 2. Arbyn M, Xu L, Simoens C, Martin-Hirsch PP. Prophylactic vaccination against human papillomaviruses to prevent cervical cancer and its precursors. Cochrane Database Syst Rev. 2018;5(CD009069). doi: 10.1002/14651858.CD009069 2. Arbyn M, Xu L, Simoens C, Martin-Hirsch PP. Prophylactic vaccination against human papillomaviruses to prevent cervical cancer and its precursors. Cochrane Database Syst Rev. 2018;5(CD009069). doi: 10.1002/14651858.CD009069 3. American Cancer Society. Lifetime Risk of Developing or Dying From Cancer. [Internet]. 2018. [accessed 30 nov. 2018]. Available at: https://www.cancer.org/cancer/cancer-basics/lifetime- probability-of-developing-or-dying-from-cancer.html 4. Unger E, Dillner J. Human Papillomavirus Laboratory Manual. 1st Edition. Geneva; 2010. 4. Unger E, Dillner J. Human Papillomavirus Laboratory Manual. 1st Edition. Geneva; 2010. 5. World Health Organization. Immunization Vaccines and Biologicals [Internet]. 2009. Available at: https://www.who.int/ immunization/research/en/ 5. World Health Organization. Immunization Vaccines and Biologicals [Internet]. 2009. Available at: https://www.who.int/ immunization/research/en/ 6. Thompson SG, Sharp SJ. Explaining heterogeneity in meta- analysis: a comparison of methods. Statistics in Medicine 1999;18:2693-708. doi: 10.1002/(SICI)1097-0258(19991030) 6. Thompson SG, Sharp SJ. Explaining heterogeneity in meta- analysis: a comparison of methods. Statistics in Medicine 1999;18:2693-708. doi: 10.1002/(SICI)1097-0258(19991030) 7. Sharp S. Meta-analysis regression. Statistics Technical Bulletin 1998;7(48):148-55. 7. Sharp S. Meta-analysis regression. Statistics Technical Bulletin 1998;7(48):148-55. 8 GoodPx. [Internet]. 2018. [ accessed 1st dez. 2018]. Available at: https://www.goodrx.com/gardasil-9?drug-name=gardasil+9 8 GoodPx. [Internet]. 2018. [ accessed 1st dez. 2018]. Available at: https://www.goodrx.com/gardasil-9?drug-name=gardasil+9 115 J. Évid-Based Healthc., Salvador, 2019 December;1(2):112-115 Doi: 10.17267/2675-021Xevidence.v1i2.2510 \| ISSN: 2675-021X J. Évid-Based Healthc., Salvador, 2019 December;1(2):112-115 Doi: 10.17267/2675-021Xevidence.v1i2.2510 \| ISSN: 2675-021X
W4297129724.txt	https://pubs.rsc.org/en/content/articlepdf/2022/sc/d2sc04661j	en		Thiazole fused <i>S</i>,<i>N</i>-heteroacene step-ladder polymeric semiconductors for organic transistors	Chemical science	2,022	cc-by	7,725	Chemical Science rsc.li/chemical-science ISSN 2041-6539 EDGE ARTICLE Mohammed Al-Hashimi et al. Thiazole fused S,N-heteroacene step-ladder polymeric semiconductors for organic transistors Volume 13 Number 41 7 November 2022 Pages 11953–12246 Chemical Science View Article Online Open Access Article. Published on 26 September 2022. Downloaded on 5/21/2024 7:17:01 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. EDGE ARTICLE Cite this: Chem. Sci., 2022, 13, 12034 All publication charges for this article have been paid for by the Royal Society of Chemistry View Journal \| View Issue Thiazole fused S,N-heteroacene step-ladder polymeric semiconductors for organic transistors† Salahuddin Attar,a Rui Yang,b Zhihui Chen,b Xiaozhou Ji, cd Marc Comı́,a Sarbajit Banerjee, c Lei Fang, c Yao Liu b and Mohammed Al-Hashimi a Ladder-type thiazole-fused S,N-heteroacenes with an extended p-conjugation consisting of six (SN6-Tz) and nine (SN9-Tz) fused aromatic rings have been synthesized and fully characterized. To date, the synthesis of well-deﬁned fused building blocks and polymers of p-conjugated organic compounds based on the thiazole moiety is a considerable synthetic challenge, due to the diﬃculty in their synthesis. Acceptor–donor building blocks M1 and M2 were successfully polymerized into ladder homopolymers P1–P2 and further copolymerized with a diketopyrrolopyrrole unit to aﬀord step-ladder copolymer P3. The optical, electronic, and thermal properties, in addition to their charge transport behavior in organic thin-ﬁlm transistors (OTFTs), were investigated. The results showed an interesting eﬀect on the molecular arrangement of the thiazole-based ladder-type heteroacene in the crystal structure revealing Received 21st August 2022 Accepted 5th September 2022 skewed p–p-stacking, and expected to possess better p-type semiconducting performance. The polymers all possess good molecular weights and excellent thermal properties. All the polymer-based DOI: 10.1039/d2sc04661j OTFT devices exhibit annealing temperature dependent performance, and among the polymers P3 rsc.li/chemical-science exhibits the highest mobility of 0.05 cm2 V1 s1. Introduction In recent years, there has been a signicant drive towards changes in the molecular design and device engineering of semiconductors to optimize their charge-carrier mobilities.1–3 Ideally, high mobility can be achieved when polymer units orient themselves in a coplanar conguration favourable for charge delocalization along a p-conjugated backbone.4,5 Conjugated ladder-type small molecules and macromolecules, which feature coplanar and rigid p-conjugated backbones, have emerged as an intriguing class of new organic materials, due to their unique electrical, physical and chemical properties.6–11 In contrast to conventional conjugated materials which tend to adopt a non-zero dihedral angle conformation as a result of thermal uctuation or torsional strain,12 the fused rings in a pconjugated ladder-type backbone have a low degree of bond rotation, thus leading to a linear and torsion-free planar conformation. As a result, this leads to the reduction of the re- a Department of Chemistry, Texas A&M University at Qatar, P.O. Box 23874, Doha, Qatar. E-mail: mohammed.al-hashimi@tamu.edu b Beijing Advanced Innovation Center for So Matter Science and Engineering, Beijing University of Chemical Technology, Beijing 100029, China c Department of Chemistry, Texas A&M University, College Station 77843-3255, Texas, USA d Department of Chemical Engineering, Stanford University, Stanford 94305, California, USA † CCDC 2094684, 2096255 and 2096256. For crystallographic data in CIF or other electronic format see https://doi.org/10.1039/d2sc04661j 12034 \| Chem. Sci., 2022, 13, 12034–12044 organizational energy during charge transfer and promotes p–p electron delocalization, enabling materials with higher chargecarrier mobilities in the condensed phase.13–15 Several examples of such conjugated ladder-type copolymers with impressive high mobilities are composed of donor or acceptors units including pentacyclic indacenodithiophene (IDT),16–19 indacenodiselenophene (IDSe),20 indacenodithiazole (IDTz),21 and bithiophene imide (BTI).22 Another interesting class of fused ladder-type building blocks with promising electronic and optical properties are based on heteroacenes.23–34 Among the various reported building blocks, S,N-heteroacenes consisting of fused thiophene and pyrrole rings, a structural analogue of the electrondonating dithieno[3,2-b:20 ,30 -d]pyrrole (DTP), unit are of particular interest.35–38 Introducing solubilizing substituents on the sp2-hybridized nitrogen atom of the pyrrole moiety, enhancing the intramolecular charge transfer (ICT) interactions, and tuning the energy levels are promising strategies for inuencing the properties required to achieve high performance organic thin-lm transistors (OTFTs). Mitsudo et al. reported the rst example of a fused aromatic S,N-heteroacene (SN5) consisting of ve membered-rings. The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) energy levels of the small molecules were tuned by the functional groups on the nitrogen atoms.39 Extending the structure to six-fused aromatic rings aﬀorded S,N-heterohexacene (SN6) (Fig. 1), which was reported by Bäuerle and co-workers.40 Vacuum-deposited lms of the © 2022 The Author(s). Published by the Royal Society of Chemistry View Article Online Open Access Article. Published on 26 September 2022. Downloaded on 5/21/2024 7:17:01 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Edge Article Chemical structures of relevant PBTz, DTP, and S,N-heteroacene ladder building blocks and the newly synthesized thiazole fused SN6-Tz and SN9-Tz ladder moieties. Fig. 1 acceptor-capped SN6 oligomers exhibited a mobility of 0.021 cm2 V1 s1. The preparation of S,N-heteroacenes was further extended from SN8 to a stable SN13 tridecamer, providing data with an interesting structure–property relationship.41,42 Most recently, Wong and co-workers reported the synthesis of donor–acceptor (D–A) alternating copolymers containing pentacyclic S,N-heteroacene building blocks. OTFT devices Scheme 1 Chemical Science fabricated using the step-ladder copolymer exhibited a hole mobility of 0.1 cm2 V s1.43 However, all current reported examples are based on S,Nheteroacenes consisting of fused thiophene and pyrrole units in the backbone. Replacing the anked thiophene units with the more electron-decient thiazole moieties is a promising approach to increase the ionization potential and improve the oxidative stability. Such an example as depicted in Fig. 1 includes fused pyrrolo[3,2-d:4,5-d0 ]-bisthiazole (PBTz).34,44–46 In addition, the anking thiazole units can provide potential anchoring points for the formation of non-covalent bonds or can enhance the van der Waals interaction with neighbouring units to further rigidify and coplanarize the conjugated system.47 Until now there have been no reported examples of thiazole-fused S,N-heteroacene building blocks, which can be attributed to the diﬃculty in their synthesis. Herein, we report the rst example of ladder-type thiazolefused S,N-heteroacenes with an extended p-conjugation in the backbones consisting of six and nine fused aromatic rings (Fig. 1). The acceptor–donor building blocks were successfully polymerized into ladder homopolymers and copolymerized with a diketopyrrolopyrrole (DPP) unit to aﬀord a step-ladder copolymer. Their optical, electronic, and thermal properties and charge transport behavior in OTFTs were investigated. The results showed an eﬀect on the molecular arrangement of the thiazole-based ladder-type heteroacene in the crystal structure Synthesis of SN6 heteroacene monomers M1 and M2 and SN9 monomers 8a and b. © 2022 The Author(s). Published by the Royal Society of Chemistry Chem. Sci., 2022, 13, 12034–12044 \| 12035 View Article Online Chemical Science revealing a skewed p–p-stacking at a higher order. The polymerbased OTFT devices exhibit annealing temperature dependent performance with the highest mobility of 0.05 cm2 V1 s1 obtained for P3. Results and discussion Open Access Article. Published on 26 September 2022. Downloaded on 5/21/2024 7:17:01 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Monomer synthesis The synthetic route to monomers M1 and M2 is depicted in Scheme 1. Stille coupling of 2-(triisopropylsilyl)-5(trimethylstannyl)thiazole with 2,5-dibromothieno[3,2-b]thiophene 1 aﬀorded 2,5-bis(2-(triisopropylsilyl)thiazol-5-yl)thieno [3,2-b]thiophene 2 as an oﬀ-yellow solid in 95% yield. The structure of 2 was conrmed using single crystal X-ray diﬀraction (XRD) (Fig. S1),† and the bond lengths and torsion angles are summarized in Tables S1 and S3.† The subsequent bromination of compound 2 using excess 1,3-dibromo-5,5dimethylhydantoin (DBDMH) yielded compound 3 as a bright yellow solid. In addition, the reaction also aﬀorded 4 in small amounts as a side product. Fig. 2 Postulated mechanism for the formation of monomer 4. Edge Article We postulate that the formation of 4 can be a result of a radical initiation that takes place by the homolytic ssion of monomer 3 into R1 and R2 radicals (Fig. 2). The radical R1 then can either recombine with R2 to generate monomer 3 or react with itself to form 4, and this was only conrmed once the sample was characterized using single crystal XRD analysis (Fig. 3a,Tables S4 and S5).† In addition, the homo-dimer of R2 can also be observed in small traces as evaluated by NMR spectroscopy. Buchwald–Hartwig amination of the crude precursor 3 containing trace amounts of 4 (used without further purications) with hexadecyl amine or 2-n-octyl-1-dodecylamine in a sealed microwave vial at 100 C in the presence of tris(dibenzylideneacetone)dipalladium(0) as the catalyst under basic conditions aﬀorded triisopropylsilyl (TIPS) protected six membered fused ladder monomers 6a and 6b, and nine-membered ladder monomers 5a and 5b from precursor 4. Subsequently, TIPS deprotection of monomers 5a/b and 6a/b with TBAF aﬀorded 7a/b and 8a/b in good overall yield. Initial attempts to dibrominate monomers 7a and 7b using N-bromosuccinimide (NBS) at room temperature did not yield the desired products. Successful bromination using NBS at a lower temperature (15 C) in anhydrous chloroform aﬀorded the desired monomers M1 and M2 (Schemes 1 and 2) in 94% and 82% yield, respectively. Interestingly the six thiazole fused S,N-heteroacene monomer 7b exhibits a blue emission in chloroform solution under 365 nm UV light, while the nine-heterocyclic ring system 8b exhibits green emission (Scheme 1), which can be attributed to the extension of the heterocyclic ring system. Single crystal XRD analysis conrmed the structure of the hexadecyl ladder monomer 7a (Fig. 3b, Tables S6 and S7),† which crystallizes in the monoclinic crystal system, exhibiting a centrosymmetric Molecular structures of (a) monomer 4; (b) 7a (R ¼ n-hexadecyl) with thermal ellipsoids drawn at the 50% probability level; (c) crystal packing of 7a along the b-axis; (d) packing of 7a showing the distance of S–S interactions (displayed in turquoise blue), and the stacking distance (shown in red) where H atoms are omitted for clarity. Fig. 3 12036 \| Chem. Sci., 2022, 13, 12034–12044 © 2022 The Author(s). Published by the Royal Society of Chemistry View Article Online Open Access Article. Published on 26 September 2022. Downloaded on 5/21/2024 7:17:01 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Edge Article Scheme 2 Synthesis of homopolymers P1 and P2 and copolymer P3. space group of P21/c at the point of inversion. In comparison to the thiophene-fused SN6 single crystal structure reported by Bäuerle,40 which has two equivalent molecules in the unit cell, the thiazole-fused 7a also exhibits two equivalent molecules in the unit cell, with a 2-fold screw axis of symmetry; a ¼ 25.4850(5), b ¼ 14.7657(3), and c ¼ 5.59660(10) Å; a ¼ 90.00, b ¼ 94.742 (2), and g ¼ 90.00 (additional information such as bond lengths and torsion angles is summarized in Tables S6 and S7).† Interestingly, the ordered non-covalent self-assembly established in the crystal packing of the thiophene based system SN6 was reported to be 3.37 Å, with a p–p interaction at a distance of 3.55 Å.40 However, the thiazole containing moiety 7a along the caxis through the anking thiazole sulfur–sulfur (S–S) interactions was measured to have a slightly shorter contact of 3.35 Å, which is lower than the sum of van der Waals radii (1.8 Å for one S) (Fig. 3d). The molecular arrangement of 7a in the crystal structure reveals a skewed p–p stacking with a distance of 3.63 Å in the columns. The bond angle of S–C–N in the thiazole moiety is 116.6 , while for the thiophene fused SN6 it was reported to be 113.8 which is slightly lower. The presence of the nitrogen atom in the thiazole moiety also has an eﬀect on the bond length (1.305 Å) in comparison to the thiophene counterpart (1.366 Å) in SN6. Intermolecular charge transfer is closely related to the orientation and molecular packing in the backbone. Based on the single crystal structure of 7a, charge transfer integrals between two p–p stacked molecules were calculated. A large hole charge transfer integral between two molecules is found at 0.27 eV, which indicates eﬃcient hole transfer in the single crystal. However, the electron transfer is less eﬃcient with a transfer integral at 0.13 eV, and thus, the polymers derived from thiazole-fused S,N-heteroacenes are expected to possess better p-type semiconducting performance. The good intermolecular charge transfer could be attributed to the co-facial orientation between molecules, which is promoted by the ladder-type backbone structure. As a comparison, the calculated hole and electron charge transfer integrals in compound 2 are lower (0.08 and 0.03 eV) than that of 7a, 0.27 and 0.12 eV, respectively. This can be attributed to the twisted stacking orientation present in the molecule (Fig. S2 and Table S8†). Chemical Science palladium(0). Copolymer P3 was synthesized by copolymerization of monomer M1 with the stannylated DPP monomer. All the polymers were precipitated in acidied methanol and puried via Soxhlet extraction with a sequence of reuxing methanol, acetone, and n-hexane. Finally reuxing chloroform was used to extract the polymers. Aer removing the solvent, homopolymers P1 and P2 were isolated as dark blue solids, while the copolymer P3 was isolated as a dark green solid. All the polymers possessed good solubility in common chlorinated solvents such as chloroform and chlorobenzene. The number-average molecular weight (Mn) and the polydispersity index (Đ) were determined via gel permeation chromatography (GPC) in chlorobenzene solution at 85 C using polystyrene standards as the calibrants. The Mn of homopolymers P1 and P2 was measured to be 11.7 and 13.6 kDa, respectively, with a narrow Đ in the range of 1.8–2.0, while copolymer P3 exhibited an Mn of 20.1 kDa with a Đ of 2.1. Thermogravimetric analysis (TGA) and diﬀerential scanning calorimetry (DSC) under an inert atmosphere were used to investigated the thermal properties of polymers P1–P3 (Fig. S3a and b).† The thermal decomposition temperatures (Td) at 5% weight loss for the three polymers P1–P3 were measured to be above 300 C. Homopolymers P1 and P2 having diﬀerent alkyl side chains (linear vs. branched) and the copolymer P3 presented similar features with the onset weight loss corresponding to the elimination of alkyl side chains. The DSC data did not reveal any pronounced thermal transitions in the range of 25 to 300 C, as a result of the low degree of order in the polymer thin lm. Optical and electrochemical properties The optical properties of the novel thiazole fused heteroacene SN6-Tz 7b and SN9-Tz 8b monomers were investigated using Ultraviolet-visible (UV-vis) and uorescence spectroscopy as depicted in Fig. 4 and summarized in Table 1. Typically, multiple vibronic splitting absorption bands are observed in S,N-heteroacenes, and this is due to the rigidity of the highly ordered, planar and fused molecular p-backbone. SN6-Tz (7b) exhibits vibronically resolved absorption bands (Fig. 4a), at lmax ¼ 334 and 321 nm corresponding to p–p electronic transitions perpendicular to the molecular axis and an energy band at lmax ¼ 371 and 390 nm attributed to the p–p* electronic transitions along the molecular axis. The lower energy transitions correspond to the HOMO–LUMO energy gap in the conjugated psystem.42,48 In comparison, the thiophene fused SN6 monomer Polymer synthesis As depicted in Scheme 2 homopolymers P1 and P2 were prepared via microwave-assisted Stille coupling of M1 and M2 with bis(tributyltin) in xylene using tetrakis(triphenylphosphine) © 2022 The Author(s). Published by the Royal Society of Chemistry Fig. 4 (a) Normalized UV-vis absorption and (b) emission spectra of SN6-Tz oligomer 7b (blue) and SN9-Tz oligomer 8b (green) in chloroform solution. Chem. Sci., 2022, 13, 12034–12044 \| 12037 View Article Online Chemical Science Edge Article Optical properties of thiazole fused S,N-heteroacene monomers 7b and 8b determined by UV-vis absorption and emission spectroscopy Open Access Article. Published on 26 September 2022. Downloaded on 5/21/2024 7:17:01 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Table 1 S,N-Heteroacene lmax (Abs)a [nm] lmax (Em)a [nm] Stokes shi (DEs)b [cm1] Egoptc [eV] EHOMOd [eV] ELUMOd [eV] SN6 SN6-Tz (7b) SN9 SN9-Tz (8b) 380 390 — 436 411 425 — 451 898 2111 — 762 3.15 3.03 — 2.70 4.85 5.19 4.66 4.85 1.24 1.58 1.53 1.76 a Measured in CHCl3. b Stokes shi-diﬀerence between the 0–0 vibronic transitions of the absorption and emission spectra. c Calculated from the low-energy onset of the absorption band. d Determined by the DFT method. exhibited energy absorption bands at lmax ¼ 362 and 380 nm. Thus, replacing the thiophene units with the thiazoles in the ladder moiety slightly lowers the optical band gap from Egopt ¼ 3.12 eV in SN6 to 3.03 eV in SN6-Tz (7b). SN6-Tz with an A–D–A architecture is expected to result in a smaller band gap in comparison to the SN6 counterpart. However, the polarized nature of the thiazole ring makes the heterocyclic thiazole unit act as both a donor and an acceptor.49 The stronger electrondonating ability of the two neighbouring substituents makes SN6-Tz a weaker D–A than expected, and as a result only a small diﬀerence of 0.12 eV is seen in the band gap. As depicted in Fig. 4a when increasing the conjugation length from six units in SN6-Tz (7b) to SN9-Tz (8b) the absorptions bands are signicantly red shied by 41–46 nm (lmax ¼ 412 and 436 nm), thus lowering the optical band gap further. Increasing the aromatic fused units from three in the DTP moiety (SN3), to ve (SN5), six (SN6), and nine (SN9) fused heterocycles, or even in the case of the thiazole moieties SN6-Tz and SN9-Tz leads to the main absorption maximum to gradually red-shi. On exciting SN6-Tz at 335 nm, a strong emission at 425 nm was observed (Fig. 4b), which is slightly red shied in comparison to the thiophene SN6 (411 nm) counterpart. The thiazole fused heteroacene SN6Tz (7b) displayed a slightly higher Stokes shi of 2111 cm1 compared to its thiophene fused SN6 counterpart (1985 cm1), due to a higher dipole moment present in the excited state as a result of the electron withdrawing eﬀect of thiazole units present at the peripherals. Similar results were observed for the extended nine membered SN9-Tz (8b) ladder monomer, exhibiting strong emission at 451 nm, with a broad shoulder at 490 nm. In addition, SN9-Tz (8b) also showed a Stokes shi of 792 cm1. The UV-vis absorption spectra of polymers P1–P3 in chloroform solution and thin lms are depicted in Fig. 5 and the optical and electrochemical properties are summarized in Table 1. Both homopolymers P1 and P2 in solution display a HOMO– LUMO absorption band represented by p–p* transitions in the visible region peaking at around 560 to 580 nm, while the n–p* energy bands are observed at around 400 nm. By incorporating the strong accepting DPP unit into the copolymer P3, the LUMO energy level is lowered and the absorption peak is red-shied to 750 nm. Going from solution to the solid state, P1 shows a slight blue shi and broadening of the maximum absorbance peak (lmax) from 561 nm to 550 nm, while P2 shows a slight red shi from 584 nm to 592 nm, indicating the diﬀerent optical behaviour of 12038 \| Chem. Sci., 2022, 13, 12034–12044 Characterization of polymers P1, P2 and P3 (a) UV-vis absorption spectra in chloroform solution; (b) as a thin ﬁlm. (c) cyclic voltammograms as a thin ﬁlm (scan rate 100 mV s1) and (d) HOMO– LUMO energy level diagram obtained using CV measurement. Fig. 5 the polymer as a result of side chain engineering. The copolymer P3 shows a blue shi from 750 to 745 nm with broadening of the absorption and a pronounced shoulder peak visible around 855 nm, and this can be attributed to aggregation with strong interchain p–p stacking, which is benecial for regular structural organization of copolymer backbones in the solid state.50–52 The optical band gaps (Egopt) of P1 and P2 calculated from the onsets of the absorption spectra in the thin lms are 1.49 and 1.51 eV, while the absorption onset of P3 is 1030 nm, resulting in a lower Egopt of 1.22 eV. Cyclic voltammetry (CV) was used to measure the oxidation and reduction potentials of polymers P1P3 in anhydrous acetonitrile under a nitrogen atmosphere. The HOMO and LUMO energy levels of the polymers were calculated using reduction and oxidation peaks with onset potentials relative to the ferrocene/ferrocenium (Fc/Fc+) redox potential as shown in Fig. 5c. All the polymers show distinct oxidation and reduction bands indicating both electron donating and electron accepting characteristics. Homopolymers P1 and P2 exhibit distinct quasi-reversible oxidation bands with onset potentials (Eox) at 0.51 and 0.49 V and an irreversible reduction band with onset potentials (Ered) at 0.84 and 0.83 V, respectively. The calculated HOMO/LUMO energy © 2022 The Author(s). Published by the Royal Society of Chemistry View Article Online Edge Article Table 2 Parameters of ﬁeld-eﬀect transistor devices based on polymers P1–P3. Top-gate/bottom contact (TG/BC) Material Method mave (cm2 V1 s1)a mmax (cm2 V1 s1)a Vth (V) IOn/IOﬀ P1 RT 200 C 250 C 300 C RT 120 C 180 C 200 C RT 120 C 180 C 200 C 200 C 250 C 300 C 1.6 104 1.3 103 2.2 104 1.2 104 6.1 105 7.7 105 3.0 105 2.0 104 1.4 104 8.2 104 1.2 103 3.0 103 0.04 1.7 103 6.4 104 3.2 104 3.1 103 2.7 104 2.0 104 1.3 104 2.5 104 6.6 105 4.5 104 2.2 104 1.6 103 2.4 103 3.3 103 0.05 2.2 103 7.7 104 11/7 3/8 9/16 12/16 4/8 9/17 14/16 8/15 20/23 25/29 14/26 21/27 23/27 23/28 23/28 10 103 103 102 102 103 103 103 103 103 103 104 103 104 103 P2 Open Access Article. Published on 26 September 2022. Downloaded on 5/21/2024 7:17:01 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Chemical Science P3 a Determined from the following equation in the saturation region; IDS ¼ (W/2L) Cim (VGS – Vth)2. levels of P1 and P2 are 4.85/3.50 eV and 4.83/3.51 eV. In comparison, copolymer P3 presented strong irreversible oxidation and reduction bands, with Eox/Ered at 0.47/0.81 V, which correspond to HOMO/LUMO energy levels at 4.81/3.53 eV. The estimated electrochemical band gaps, Eel g , of polymers P1– P3 are in the range of 1.28–1.35 eV and are consistent with the Egopt calculated from the UV-vis absorption spectra. DFT computational studies To evaluate the eﬀect of replacing the fused thiophene with the fused thiazole unit, a comparative DFT calculation (B3LYP/6311G+(d,p)) was performed based on the structures of SN6 and SN6-Tz. Structure optimization was performed at the same level for SN9 and SN9-Tz, while all alkyl groups were simplied with methyl groups to shorten calculation times. Compared to the thiophene end groups in SN6 and SN9, the electron withdrawing thiazole units show slightly lower electron density (Fig. 6) on the HOMO and higher electron density on the LUMO, and thus as a result decrease both HOMO and LUMO energy levels. The thiazole groups pull the electron density from the heterocyclic SN-p-system, leading to a donor–acceptor system, and thus the stabilization of the frontier molecular orbital energies is achieved with narrowing of the band gaps. The extended SN9-Tz heteroacene shows an obviously lower calculated energy band gap (3.09 eV) than SN6-Tz (3.61 eV). However, the calculated results are both slightly at a higher end than the experimental Egopt values; this could be attributed to the solvation eﬀect. OTFT charge transport characterization The charge transport properties and electrical behavior of P1– P3 polymers were analysed by fabricating bottom-gate/bottomcontact (BG/BC) OTFTs (Table 2). Gold (Au) and titanium (Ti) layers were thermally evaporated onto a SiO2 (300 nm)/p++-Si substrate using a shadow mask to form source and drain contacts, and this was followed by octadecyl trimethoxy silane (OTS) treatment. Optimized geometries of thiophene fused SN6, thiazole fused SN6-Tz, thiophene fused SN9 and thiazole fused SN9-Tz molecules and spatial electron distributions of frontier molecular orbitals (FMOs) and their energy levels. Fig. 6 © 2022 The Author(s). Published by the Royal Society of Chemistry Chem. Sci., 2022, 13, 12034–12044 \| 12039 View Article Online Open Access Article. Published on 26 September 2022. Downloaded on 5/21/2024 7:17:01 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Chemical Science Edge Article Fig. 7 (a) Transfer and (b) output characteristics of the best OTFT devices based on polymers P1, P2, and P3 under optimal conditions. The polymer solutions were prepared with the same concentration of 5 mg mL1 in chloroform and were coated on the OTS-treated substrate to form organic semiconducting layers in a N2-lled glovebox using spin-coating and then annealed at diﬀerent temperatures for 15 min. The typical output and transport characteristics for the devices based on P1–P3 are depicted in Fig. 7 and ESI.† The charge carrier mobilities (m) and the threshold voltage (Vth) were extracted from the saturated region in the transfer curves. The OTFT optimal polymer device performance is summarized in Table 3. The initial fabrication of BG/BC devices at various annealing temperatures in order to optimize the charge mobility of polymers P1–P3 was carried out. Generally, thermal annealing represents a satisfactory strategy to optimize the device performance via improving lm formation and promoting the “edge on” orientation and crystallinity of polymer chains, which are believed to benet the charge mobility.53,54 Interestingly, the mobility performance of all polymers exhibits annealingtemperature dependence up to 200 C, with better improvements in the transistor performance for homopolymer P1 and copolymer P3, which contain a straight chain substituted ladder unit. This indicates that having a linear alkyl substituent on the pyrrole fused ring can improve the polymer skeleton arrangement with increasing annealing temperature.55 Without annealing, the devices fabricated for the homopolymers Table 3 Crystallinity and morphology Thin lms of polymers P1–P3 were fabricated and measured using XRD in order to analyse the relationship between the chemical structure of the polymers and their crystallinity behaviour, and the lamellar/p–p stacking d-spacing values are summarized in Table 2. As depicted in Fig. 8, all polymers exhibit a similar degree of crystallinity with lamellar 2q peaks in the range 3.7 to 4.6 and p–p stacking peaks ranging from 21 to 24 . With the branched side chains, P2 presented a larger lamellar packing distance (23.86 Å) compared to P1 (21.53 Å) and P3 (19.19 Å), which possess linear side chains. Similarly, the p–p stacking distance of P2 (4.50 Å) is also larger than that of P1 (3.77 Å) and P3 (3.69 Å). In addition, small lamellar reection at 2q ¼ 7.2 was observed for P3. These results suggest that copolymer P3 presents better packing and higher crystallinity than homopolymers P1 and P2, which is in good alignment with the solid-state UV-vis absorption data and as a result could contribute to higher mobility performance in OTFT devices. Additionally, morphological properties in the solid-state thin lms for P1–P3 were evaluated using atomic force microscopy (AFM). As shown in Fig. 9 the AFM images of P1 and P3 with linear side chains present slightly better homogeneity, in comparison with P2, which exhibits some nonuniform regions Summary of the molecular weights, thermal, optical, electrochemical, XRD and AFM parameters of polymers P1–P3 Mna (kDa) Đ P1 12 P2 14 P3 20 displayed a maximum charge mobility of 3.2 104 cm2 V1 s1 for P1 and 1.3 104 cm2 V1 s1 for P2, while copolymer P3 presented a maximum mobility of 2.2 104 cm2 V1 s1. Aer thermal annealing at 200 C for 15 min, the mobilities were improved by one order of magnitude for the linear alkyl substituted ladder unit-based polymers P1 and P3, with charge mobilities ranging from 3.1–3.3 103 cm2 V1 s1. Devices fabricated from homopolymer P2 exhibit discrete uctuations of charge mobility with increasing thermal annealing temperatures with the best performance of 4.5 104 cm2 V1 s1 obtained at 200 C. In addition, thermal annealing at higher temperatures of 250 and 300 C for devices based on P1 and P3 resulted in lower charge carrier mobility by one order of magnitude of 2.0 104 and 7.7 104 cm2 V1 s1. We further attempted to enhance the OTFT performance of copolymer56 P3 by exchanging the conguration of the device to topgate/bottom contact (TG/BC). Aer annealing at 200 C, P3 displayed an improved mobility of 0.05 cm2 V1 s1 and a Vth of 23 V, with an on/oﬀ current ratio of 103. Meanwhile no signicant change was observed for the other polymers P1 and P2 using the TG/BC device architecture. e optf Td ( C) lmaxsolb (nm) lmaxlmc (nm) HOMOd (eV) LUMOd (eV) (Eel (eV) Lamellar ddd,g (Å) p–p ddd,g (Å) RMS (nm) g ) (eV) Eg 1.8 314 2.0 324 2.1 326 561 584 750 550 592 745 4.85 4.83 4.81 3.50 3.51 3.53 1.35 1.32 1.28 1.51 1.49 1.22 21.53 23.86 19.19 3.77 4.50 3.69 0.80 1.04 0.75 a Determined by GPC (against polystyrene standards) in chlorobenzene at 85 C. b lmax in chlorobenzene solution. c Spin-coated from chlorobenzene solution onto a glass surface. d EHOMO/ELUMO ¼ [(Eonset Eonset (FC/FC+ vs. Ag/Ag+)) 4.8] eV, where 4.8 eV is the energy level of ferrocene below the vacuum level and the formal potential Eonset(FC/FC+ vs. Ag/Ag+) is equal to 0.45 V. e Electrochemical bandgap: Eel g ¼ Eox/ f opt ¼ 1240/ledge. g Calculated by using Bragg's Law d ¼ l/(2 sin q), where l ¼ 1.5406 Å. (—) Non-observable. onset Ered/onset. Optical bandgap: Eg 12040 \| Chem. Sci., 2022, 13, 12034–12044 © 2022 The Author(s). Published by the Royal Society of Chemistry View Article Online Open Access Article. Published on 26 September 2022. Downloaded on 5/21/2024 7:17:01 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Edge Article Fig. 8 XRD patterns acquired for copolymers P1–P3. Chemical Science a slightly enhanced coplanarity in the backbone. In addition, SN6-Tz exhibits highly structured and vibronically resolved absorption bands, which are red-shied in comparison to the thiophene fused SN6 monomers exhibiting slightly lower absorption bands. Thus, replacing the thiophene units with the thiazoles in the ladder moiety slightly lowers the optical band gap from 3.12 eV to 3.03 eV. When increasing the conjugation length from six units in the thiazole ladder monomer to nine, the absorptions bands are signicantly red shied by 41–46 nm, thus further lowering the optical band gap. Furthermore, the thiazole-fused heteroacene SN6-Tz displayed a slightly higher Stokes shi compared to its thiophene fused SN6 counterpart. Ladder homopolymers P1 and P2, and step-ladder copolymer P3 constructed from thiazole-fused S,N-heteroacene moieties present good solubility and high thermal stability up to 300 C. All the polymers exhibit narrow band gaps ranging from 1.28 and 1.35 eV. The presence of the strong accepting DPP unit in the copolymer P3 backbone increases the p–p interactions and promotes the packing of polymer chains in the solid-state, which was investigated by thin-lm absorption and XRD characterization. The absence of the DPP unit in the homopolymers P1 and P2 results in lower OTFT mobilities in the order of 103 and 104 cm2 V1 s1, respectively. Meanwhile P3 exhibits mobility values as high as 0.05 cm2 V1 s1. Overall, this study has demonstrated that step-ladder polymers consisting of thiazole-fused S,N-heteroacene building blocks can be suitable candidates for the fabrication of semiconductor OTFTs. Data availability The datasets supporting this article have been uploaded as part of the ESI material.† Fig. 9 Tapping-mode AFM images of (a) the height, (b) phase with a 0.4 mm 0.4 mm scan size and (c) 3D-topography of copolymers P1–P3. on the surface (Fig. 9a). P2 has a rougher surface with a root mean square (RMS) roughness of 1.04 nm, while P1 and P3 show slightly lower values of 0.80 and 0.75 nm, respectively. In addition, P2 presents bigger grain sizes as depicted in the phase image of Fig. 9b, in comparison with P1 and P3. These small diﬀerences were easily detected by 3D-height topography as shown in Fig. 9c (represented by the same scale for the polymers). Author contributions S. Attar carried out the synthesis; R. Yang, Z. Chen & Y. Liu carried out OFET measurements; X. Ji did the DFT computational studies; M. Comi XRD and AFM measurements; S. Banerjee, and L. Fang edited and contributed in the writing of the manuscript with M. Al-Hashimi taking the led in with idea and nialzing. Conﬂicts of interest There are no conicts of interests to declare. Conclusion Acknowledgements In summary, we have reported the design, synthesis and characterization of four novel S,N-heteroacene based thiazole endfused ladder monomers. Linear and branched alkyl groups on the nitrogen atoms aﬀorded six fused aromatic ladder-type molecules M1 and M2. We further extended the conjugation length to aﬀord ladder-type 5a–b with nine-fused rings in excellent solubility. In comparison to the thiophene-fused analogue SN6, the thiazole-fused molecules show a diﬀerent molecular arrangement in the crystal structure and have The authors gratefully acknowledge the support of this work from the Qatar National Research Found (QNRF) and the National Priorities Research Program, project number NPRP11S-1204-170062. © 2022 The Author(s). Published by the Royal Society of Chemistry Notes and references 1 H. Bronstein, C. B. Nielsen, B. C. Schroeder and I. McCulloch, The role of chemical design in the Chem. Sci., 2022, 13, 12034–12044 \| 12041 View Article Online Chemical Science Open Access Article. Published on 26 September 2022. Downloaded on 5/21/2024 7:17:01 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 2 3 4 5 6 7 8 9 10 11 12 13 performance of organic semiconductors, Nat. Rev. Chem., 2020, 4(2), 66–77. Y. Zhou, T. Kurosawa, W. Ma, Y. Guo, L. Fang, K. Vandewal, Y. Diao, C. Wang, Q. Yan, J. Reinspach, J. Mei, A. L. 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https://openalex.org/W2152291864	https://hal.science/hal-01250502/file/Brunner-PS-2013-OA_%7BFDD8BD65-8F45-4E75-A623-AEE24849AEB5%7D.pdf	English	null	Fine-root turnover rates of European forests revisited: an analysis of data from sequential coring and ingrowth cores	Plant and soil	2,012	cc-by	10,331	Plant Soil (2013) 362:357–372 DOI 10.1007/s11104-012-1313-5 Plant Soil (2013) 362:357–372 DOI 10.1007/s11104-012-1313-5 REVIEW ARTICLE Fine-root turnover rates of European forests revisited: an analysis of data from sequential coring and ingrowth cores I. Brunner & M. R. Bakker & R. G. Björk & Y. Hirano & M. Lukac & X. Aranda & I. Børja & T. D. Eldhuset & H. S. Helmisaari & C. Jourdan & B. Konôpka & B. C. López & C. Miguel Pérez & H. Persson & I. Ostonen I. Brunner & M. R. Bakker & R. G. Björk & Y. Hirano & M. Lukac & X. Aranda & I. Børja & T. D. Eldhuset & H. S. Helmisaari & C. Jourdan & B. Konôpka & B. C. López & C. Miguel Pérez & H. Persson & I. Ostonen I. Brunner & M. R. Bakker & R. G. Björk & Y. Hirano & M. Lukac & X. Aranda & I. Børja & T. D. Eldhuset & H. S. Helmisaari & C. Jourdan & B. Konôpka & B. C. López & C. Miguel Pérez & H. Persson & I. Ostonen Received: 23 December 2011 /Accepted: 28 May 2012 /Published online: 8 July 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com rates of common European forest tree species and to compare them with most frequently published values. Methods We compiled available European data and applied various turnover rate calculation methods to Abstract Background and Aims Forest trees directly contribute to carbon cycling in forest soils through the turnover of their fine roots. In this study we aimed to calculate root turnover Responsible Editor: Alexia Stokes. Responsible Editor: Alexia Stokes. Responsible Editor: Alexia Stokes. I. Brunner (*) Swiss Federal Institute for Forest, Snow and Landscape Research WSL, 8903 Birmensdorf, Switzerland e-mail: ivano.brunner@wsl.ch X. Aranda: C. Miguel Pérez Ecophysiology, IRTA Torre Marimon, 08140 Caldes de Montbui, Spain I. Børja: T. D. Eldhuset Norwegian Forest and Landscape Institute, P.O. Box 115, 1431 Ås, Norway M. R. Bakker University of Bordeaux, UMR1220 TCEM, 33175 Gradignan, France H. S. Helmisaari Department of Forest Sciences, University of Helsinki, P.O. Box 27, 00014 Helsinki, Finland C. Jourdan CIRAD, UMR Eco&Sols, 2, Place Pierre Viala, 34060 Montpellier, France C. Jourdan CIRAD, UMR Eco&Sols, 2, Place Pierre Viala, 34060 Montpellier, France B. Konôpka Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, Kamýcka 129, Suchdol, 16521 Prague, Czech Republic B. Konôpka Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, Kamýcka 129, Suchdol, 16521 Prague, Czech Republic 358 Plant Soil (2013) 362:357–372 NPP Net primary production P Production SOM Soil organic matter T Turnover rate the resulting database. We used Decision Matrix and Maximum-Minimum formula as suggested in the literature. Results Mean turnover rates obtained by the combina- tion of sequential coring and Decision Matrix were 0.86 yr−1 for Fagus sylvatica and 0.88 yr−1 for Picea abies when maximum biomass data were used for the calculation, and 1.11 yr−1 for both species when mean biomass data were used. Using mean biomass rather than maximum resulted in about 30 % higher values of root turnover. Using the Decision Matrix to calculate turnover rate doubled the rates when compared to the Maximum-Minimum formula. The Decision Matrix, however, makes use of more input information than the Maximum-Minimum formula. Introduction Turnover of tree fine roots is one of the major carbon (C) pathways in forests. The cause of the large C flux through this biomass pool is the rather limited lifespan of tree roots less than 2 mm in diameter. Given the estimated size of the C flux associated with the limited lifespan (synonyms: ‘longevity’ or ‘turnover time’, in- verse of ‘turnover rate’) of fine roots, thought to reach 0.5 to 3 tC ha−1 yr−1 in steady-state forest ecosystems (Gill and Jackson 2000; Brunner and Godbold 2007), we clearly need to have a good understanding of the turnover rate at which fine roots die and contribute to soil C pools. Indeed, in the light of ongoing and pro- jected climate change and the implementation of C reporting in many countries, belowground C dynamics have to be taken into account. Given the role of scientists in this debate, it is down to those who study root dynam- ics to provide the knowledge basis that permits modellers and C reporters to utilise the most realistic turnover values. Currently, root turnover rates are commonly uti- lised to parameterise biogeochemical models, which re- quire fine root turnover rate data input e.g. Biome-BGC, LPJ, or LPJ-GUESS (e.g. Pietsch et al. 2005; Sitch et al. 2003; Smith et al. 2001). The turnover rates as input can be derived from published scientific literature, based on supposed relationships between leaf lifespan and fine root lifespan (i.e. lower turnover rates for evergreen species with long-lived leaves) with values between 0.18 and 1.02 yr−1 (Pietsch et al. 2005; Cienciala and Tatarinov 2006; Tatarinov and Cienciala 2006) or sim- plified to just one value 0.7 yr−1 as suggested recently by Hickler et al. (2008). Using the most appropriate turnover rates will improve the capacity of these models to assess the change in belowground C pools in forests. Conclusions We propose that calculations using the De- cision Matrix with mean biomass give the most reliable estimates of root turnover rates in European forests and should preferentially be used in models and C reporting. Keywords Annual production . Decision Matrix . Fine-root turnover rates . Ingrowth cores . Maximum- Minimum formula . Sequential coring Keywords Annual production . Decision Matrix . Fine-root turnover rates . Ingrowth cores . Maximum- Minimum formula . Introduction Meanwhile, minirhizotron studies are not able to determine the exact time of root death. In addition, the installation of the minirhizotron tubes can change water and temperature regimes as well as soil matrix resistance to root penetration. Moreover, fine root growth is often stimulated by the conditions along the minirhizotron tube. Unsurprisingly, direct comparisons of these two methods result in a discrepancy in root longevity estimates (Tierney and Fahey 2002; Strand et al. 2008; Gaul et al. 2009), sometimes explained by different fractions of fine roots under observation, i.e. the short-lived and the long-lived fine roots, likely to be recorded by these two methods (Gaudinski et al. 2010). is the sequential coring technique (e.g. Stober et al. 2000; Ostonen et al. 2005). Here, several series of soil cores are sampled at defined intervals over a period of at least 1 year. Fine roots are extracted from the soil cores and the differences of the dry mass of living (biomass) and dead (necromass) fine roots between two time points recorded. Taking advantage of data generated by sequen- tial coring, several methods exist to calculate the produc- tion from the change of the fine-root biomass and necromass data. The production can be calculated by the ‘Maximum-Minimum’ formula (McClaugherty et al. 1982), by the ‘Decision Matrix’ formula (Fairley and Alexander 1985), or by the ‘Compartment Flow’ formula (Santantonio and Grace 1987). Whereas the ‘Maximum- Minimum’ formula uses only biomass data, the other two methods require both biomass and necromass data. The ‘Compartment Flow’ formula further requires decompo- sition data of fine root litter (e.g. Silver et al. 2005, Osawa and Aizawa 2012). Thus, the values of fine root turnover rates can vary not only due to measurement methods but also due to calculation methods applied (e.g. Publicover and Vogt 1993; Vogt et al. 1998; Strand et al. 2008). A true comparison of the various turnover rates may only be possible by using observations from same sites where various methods were applied (e.g. Haynes and Gower 1995; Ostonen et al. 2005; Hendricks et al. 2006; Gaul et al. 2009). As for the popularity of different measurement methods, many more estimates of root turnover rates are available from sequential coring and ingrowth cores than from the minirhizotron method (Finer et al. 2011). Introduction Sequential coring Abbreviations B Biomass BGC Biogeochemical cycles C Carbon DM Decision Matrix GPP Gross primary production GUESS General ecosystem simulator LPJ Lund-Potsdam-Jena model MM Maximum-Minimum MRT Mean residence time N Necromass B. C. López CREAF Center for Ecological Research and Forestry Applications, and Ecology Unit of the Department of Plant and Animal Biology and Ecology, Edifici Ciències UAB, 08193 Bellaterra, Spain H. Persson Department of Ecology, Swedish University of Agricultural Sciences SLU, P.O. Box 7044, 750 07 Uppsala, Sweden I. Ostonen Institute of Ecology and Earth Sciences, University of Tartu, Vanemuise 46, 51014 Tartu, Estonia Abbreviations B Biomass BGC Biogeochemical cycles C Carbon DM Decision Matrix GPP Gross primary production GUESS General ecosystem simulator LPJ Lund-Potsdam-Jena model MM Maximum-Minimum MRT Mean residence time N Necromass Fine root turnover rate is dependent on the fine root biomass and the annual production of fine roots, but also on the various methods and calculations (e.g. Jourdan et al. 2008; Gaul et al. 2009; Finer et al. 2011; Yuan and Chen 2010). However, there is quite some uncertainty regarding which fine root turnover rates would be most suitable for end users. This is illustrated by the ongoing debate among scientists about how the turnover rate of H. Persson Department of Ecology, Swedish University of Agricultural Sciences SLU, P.O. Box 7044, 750 07 Uppsala, Sweden I. Ostonen Institute of Ecology and Earth Sciences, University of Tartu, Vanemuise 46, 51014 Tartu, Estonia Plant Soil (2013) 362:357–372 359 the fine roots can be estimated best and which method is the most suitable (e.g. Strand et al. 2008; Trumbore and Gaudinski 2003; Majdi et al. 2005; Jourdan et al. 2008). Starting from the most recent developments, stable C- isotopes and radiocarbon (13C, 14C) may be used to estimate root carbon longevity, either by using labelling techniques or natural abundances in the atmosphere (e.g. Matamala et al. 2003; Gaudinski et al. 2001, 2010; Endrulat et al. 2010). A more widely used method to estimate the lifespan of fine roots is the use of minirhizo- trons (e.g. Johnson et al. 2001; Majdi and Andersson 2005). This technique allows for a direct observation of individual roots and their development. Both methods suffer from several drawbacks, the main weakness of isotopic analysis for root age determination is the uncer- tain age of organic compounds used to construct fine roots (Sah et al. 2011). Data origin The Maximum-Minimum (MM) formula calculates the annual fine-root production (Pa) by subtracting the lowest biomass (Bmin) from the highest biomass value (Bmax) irrespectively of other biomass values recorded during a full year (McClaugherty et al. 1982). Necro- mass data are not required for this method: Our study was carried out on data of fine root biomass and necromass of European forest tree species, extracted from published studies, found through regular literature research in library databases or supplied by members of our COST network. A large proportion of the data orig- inates from doctoral theses due to the availability of raw data in this type of publication. We only included datasets where data collection was carried out for at least one full year. Fine root production was measured either directly by the use of the ingrowth core method or indirectly by the use of the sequential coring method (see Ostonen et al. 2005). Fine root biomass was defined as the amount of living fine roots occurring in the soil at any given time. Sequential coring was used to establish fine root biomass in most studies, apart from the case of the ingrowth core method where biomass usually was estimated from a single coring. We did not consider data originating from minirhizotron studies as these are reviewed elsewhere (Børja et al. in preparation). Finally, the dataset created for this study included 17 studies with 31 datasets for sequential coring and 7 for ingrowth core studies. The most abundant data sets obtained by sequential coring were available for Fagus sylvatica and Picea abies with 13 and 11 data sets, respectively (Table 3). Data sets of other tree species, e.g. Pinus sylvestris, Populus spp., and Quercus spp., were present only in three or fewer data sets. More than 80 % of the data were from forests with adult trees (‘steady state’ conditions). Data sets originat- ing from ingrowth cores were available only for F. syl- vatica, P. abies, and P. sylvestris, and with only two to three data sets per tree species (Table 4). Introduction Alternatively, instead of direct observations of indi- vidual root longevity, the mean lifespan can be calculated by dividing the ‘pool’ (biomass) by its ‘input’ (annual production). Because the turnover rate is the inverse of lifespan, it can be calculated by dividing the ‘annual production’ by the ‘belowground standing crop’ (0biomass) (Gill and Jackson 2000). There are several methods used to obtain estimates of annual fine root production. A widely used method to directly measure the production of fine roots is the use of ingrowth cores (e.g. Persson 1980a, 1980b; Vogt and Persson 1991). This method measures the amount of fine roots which grow into a defined volume of root-free soil over a defined period of time. The advantage of this method is its relative ease and speed of application when estimating root production (Vogt and Persson 1991). More recently, root nets were applied instead of ingrowth cores to min- imise soil disturbance during the installation (Hirano et al. 2009; Lukac and Godbold 2010). An alternative method to indirectly measure the production of fine roots We took advantage of the European COST network FP0803 “Belowground carbon turnover in European for- ests” bringing together root researchers from 30 Europe- an countries, to investigate on the sources of variation in turnover estimates available in the literature. In particular, as our group covered most of the European research groups that have worked on fine root turnover in the last decades, we were able to reunite/mine detailed datasets needed to evaluate the effect of calculation methods on fine root turnover rates in the European context. This implies that the implications of our work are restricted to European tree species and growth conditions. Our objec- tives were 1) to evaluate the pure effect of calculation methods on mean turnover rates of European forests and their ranges; 2) to evaluate how other factors such as soil stratification contribute to the calculated/perceived vari- ation in turnover rates of European forests, and 3) to propose turnover rates and ranges for end users for the most common European forests. 360 Plant Soil (2013) 362:357–372 Materials and methods years are the minimum requirement for the calculation of root production. Data origin Pa MM ð Þ ¼ Bmax Bmin ð1Þ ð1Þ The Decision Matrix (DM) calculates the annual fine-root production (Pa) by summing all calculated productions (P) between each pair of consecutive sam- pling dates throughout a full year: Pa DM ð Þ ¼ X P ð2Þ ð2Þ Pa DM ð Þ ¼ X P The production (P) between two sampling dates is calculated either by adding the differences in biomass (ΔB) and necromass (ΔN), by adding only the differ- ences in biomass (ΔB), or by equalling P to zero (Fairley and Alexander 1985). The conditions with which of the P formulas to be used are as follows: P ¼ ΔB þ ΔN aÞ if biomass and necromass have increased bÞ if biomass has decreased and necromass has increased; but jΔBj lower than jΔNj ð3Þ ð3Þ P ¼ ΔB if biomass has increased and necromass has decreased ð4Þ ð5Þ The Decision Matrix used as the basis for calculations is shown in Table 1. To calculate the annual production, all production values from interim periods are summed up from the start of sequential coring until the same time point in the following year (see also Table 2a, b). In the present study, all differences in biomass and necromass were taken into account during the calculation, assuming that the living and dead pool are continuously changing. However, some authors suggest summing up only the statistically significant differences (e.g. Stober et al. 2000). We propose that accounting for all differences between root biomass in two sampling dates constitutes a better approach. The size (and therefore the significance) of the difference is clearly dependent on the duration of the interim period, as well as on the season. Including significantly different observations would skew the data coverage towards long-gap observations only. Calculations of fine-root production Fine root production was calculated either with the ‘Maximum-Minimum’ formula or the ‘Decision Ma- trix’. The ‘Compartment Flow’ method was not applied because decomposition data of root litter were not suffi- ciently available. As a pre-requisite of annual fine root production calculation, a single sampling campaign must have lasted at least 12 months. Studies of less than 12 months (e.g. one vegetation period) or not of required level of detail were not considered (e.g. Konôpka et al. 2005; Konôpka 2009; López et al. 2001). At least two measurements from the same month in two consecutive 361 Plant Soil (2013) 362:357–372 Calculations of fine-root turnover rates Table 1 Decision Matrix according to Fairley and Alexander (1985). (B 0 Biomass, N 0 Necromass, P 0 Production) Biomass increase Biomass decrease Necromass increase P ¼ $B þ $N P ¼ $B þ $N a or P 0 0b Necromass decrease P0ΔB P00 a if \|ΔB\| < \|ΔN\| b if \|ΔB\| > \|ΔN\| Table 1 Decision Matrix according to Fairley and Alexander (1985). (B 0 Biomass, N 0 Necromass, P 0 Production) coring data, decision matrix calculations and mean bio- mass values were taken from Hertel (1999), Richter (2007), Makkonen and Helmisaari (1999), Bakker (1999), and Ostonen et al. (2005). In the reports relative to seven sites we had fine root data separately assessed for sub diameter classes (<1 mm, 1–2 mm) and so we could do the calculations for each diameter class and compare them with the total diameter class (<2 mm). And, utilizing studies with longest data series, we ex- plored whether and how different observations lengths (1 yr, 1–2 yr, 2–3 yr), the start of the observation period (spring, summer, autumn, winter), and the number of samplings per year may influence the turnover values. Calculations of fine-root turnover rates The turnover rate TBmax of fine roots was calculated by dividing the annual fine root production (Pa) by the high- est biomass value (maximum biomass Bmax) according to Gill and Jackson (2000) (compare also Table 2c): Statistics For statistical analyses, simple linear regression and Mann–Whitney U test, the software StatView 5.0 (SAS Institute, Cary, NY, USA) was used, with the significance level of p<0.05. The data was tested for normal distri- bution and for homogeneity of variances among groups. TBmax ¼ Pa Bmax = ð6Þ ð6Þ As an alternative, the turnover rate TBmean was calcu- lated by dividing the annual fine root production (Pa) by the mean biomass (Bmean) according to McClaugherty et al. (1982) (compare also Table 2c): Bmean ¼ P B n = n ¼ number of samples per year ð Þ Bmean ¼ P B n = n ¼ number of samples per year ð Þ Bmean ¼ P B n = n ¼ number of samples per year ð Þ ð7Þ TBmean ¼ Pa Bmean = ð8Þ Fine-root turnover rate ð8Þ Turnover rates obtained by the combination of sequential coring, Decision Matrix method, and the maximum bio- mass data varied from 0.19 to 2.04 yr−1 for F. sylvatica and from 0.44 to 1.36 yr−1 for P. abies (Table 3), with mean values for F. sylvatica and P. abies of 0.86 and 0.88 yr−1, respectively (Table 5). Using the mean biomass instead of the maximum biomass, the turnover rates var- ied from 0.23 to 2.92 yr−1 for F. sylvatica and from 0.56 to 1.77 yr−1 for P. abies (Table 3), with mean values of 1.11 yr−1 for both F. sylvatica and P. abies (Table 5). For other tree species, less than three data sets were available, e.g. only 2 data sets were available for P. sylvestris, and both had turnover rates higher than 1.5 yr−1 (Table 3). Assessment of other factors generating variation in fine-root turnover rates Assessment of other factors generating variation in fine-root turnover rates Utilizing the raw datasets in our database, we analysed several other factors for their influence of fine root turnover, based on subsamples of the database for se- quential coring only. These factors included 1) soil strat- ification, 2) soil depth, 3) root diameter, 4) observation length, 5) start of observation period and 6) number of samplings per year. For the soil stratification approach we used 13 sites with detailed root data for the various soil layers. Briefly, for the layer-per-layer approach we computed fine root production per layer and summed this as a fine root production for the entire profile. Turnover rate was then computed as production divided by average fine root biomass for the entire profile. For the whole profile approach, instead, we used the summed bio- and necromass values for the entire profile to com- pute fine root production and then divided by average fine root biomass to calculate the fine root turnover rate. For this comparison of the two approaches, sequential Turnover rates obtained by the combination of se- quential coring, Maximum-Minimum method, and max- imum biomass data were consistently below 0.7 yr−1 for F. sylvatica and P. abies (Table 3), with mean turnover rates of 0.41 yr−1 and 0.44 yr−1, respectively (Table 5). The mean turnover rate of P. sylvestris was 0.48 yr−1 and did fall in a similar range (Table 5). Using the mean biomass instead of the maximum biomass, the turnover rates ranged from 0.26 to 0.95 yr−1 for F. sylvatica and P. 362 Plant Soil (2013) 362:357–372 abies (Table 3), with mean turnover rates of 0.53 yr−1 for F sylvatica and 0 57 yr−1 for P abies (Table 5) maximum biomass, the mean turnover rates were higher 2 58 1 15 and 1 40 yr−1 for F sylvatica P Table 2 Worked sample with a data set from sequential coring (data from Ostonen et al. 2005). Formula [3] P ¼ $B þ $N ð Þ and [4] (P 0 ΔB) are according to Fairley and Alexander (1985). Other formula are according to the Material and Methods sec- tion. (P 0 Production, B 0 Biomass, N 0 Necromass, T 0 Turnover rate) a) Calculation of the production P using the Decision Matrix. Assessment of other factors generating variation in fine-root turnover rates Sampling date Biomass (g m−2) Necromass (g m−2) Formula Calculation Production P (g m−2 t−1) June 1996 127 130 July 1996 161 178 [3] (161-127)+(178-130) 82 Aug. 1996 166 114 [4] 166-161 5 Sept. 1996 165 174 [3] (165-166)+(174-114) 59 Oct. 1996 199 198 [3] (199-165)+(198-174) 58 Nov. 1996 64 159 [5] 0 0 June 1997 110 125 [4] 110-64 46 Mean (±SE) [7]: 141 (±17) Sum [2]: 250 b) Calculation of the annual production Pa. Method Formula Calculation Annual production Pa (g m−2 yr−1) Decision Matrix [2] 82+5+59+58+0+46 250 Maximum-Minimum [1] 199-64 135 c) Calculation of the turnover rate T (using mean biomass Bmean or maximum biomass Bmax). Method Formula Calculation Turnover rate T (yr−1) Using Bmean Using Bmax Decision Matrix [6] 250 / 141 – 1.77 Decision Matrix [8] – 250 / 199 1.26 Maximum-Minimum [6] 135 / 141 – 0.95 Maximum-Minimum [8] – 135 / 199 0.68 a) Calculation of the production P using the Decision Matrix. abies (Table 3), with mean turnover rates of 0.53 yr−1 for F. sylvatica and 0.57 yr−1 for P. abies (Table 5). maximum biomass, the mean turnover rates were higher, 2.58, 1.15, and 1.40 yr−1 for F. sylvatica, P. abies, and P. sylvestris, respectively, using the Deci- sion Matrix, and 2.58, 0.98, and 1.31 yr−1 for F. sylvatica, P. abies, and P. sylvestris, respectively, using the Maximum-Minimum formula (Table 5). Using the ingrowth core method, in maximum three data sets were available per tree species (Table 4). Mean turnover rates obtained by ingrowth cores, the Decision Matrix method, and the maximum biomass were 1.00, 0.72, and 0.76 yr−1 for F. sylvatica, P. abies, and P. sylvestris, respectively (Table 5). Using the Maximum-Minimum method and the maximum biomass, the mean turnover rates were with 1.00, 0.62, and 0.72 yr−1, respectively, in a similar range (Table 5). Using the mean biomass instead of the We compared the difference in turnover rate estimates based on maximum or mean biomass as the denominator. On average in our dataset, using mean biomass rather than maximum resulted in about 30 % higher estimate of root turnover rate T (TBmean ¼ 1:3 TBmax 0:001; r20 0.98, p<0.001; Fig. 1). Plant Soil (2013) 362:357–372 363 Plant Soil (2013) 362:357 372 36 Table 3 Sequential coring: Mean and maximum biomass, annual production, and turnover rate of tree fine roots recorded with sequential coring. Assessment of other factors generating variation in fine-root turnover rates The annual production is calculated with the ‘Decision Matrix’ or the ‘Maximum-Minimum’ formula, and the turnover rate is calculated by dividing the annual production by the mean biomass (Bmean) or by the maximum biomass (Bmax). (B 0 Biomass) Country Site Mean annual temp. (°C) Depth (cm) Stand age (yr) Biomass (B) Decision Matrix Maximum-Minimum References Mean (g m−2) Max. (g m−2) Production Turnover rate Production Turnover rate Bmean Bmax Bmean Bmax (g m−2 yr−1) (yr−1) (yr−1) (g m−2 yr−1) (yr−1) (yr−1) Fagus sylvatica: Switzerl. Entleb. 6.7 0–25 >100 422 580 395 0.94 0.68 290 0.69 0.50 Richter (2007) Switzerl. Krauch. 8.2 0–25 >100 480 710 476 0.99 0.67 356 0.74 0.50 Richter (2007) Switzerl. Nieder. 8.7 0–25 >100 413 501 281 0.68 0.56 217 0.53 0.43 Richter (2007) Switzerl. Walter. 7.4 0–25 >100 348 441 193 0.55 0.44 171 0.49 0.39 Richter (2007) Switzerl. Vordem. 8.8 0–25 >100 807 957 597 0.74 0.62 356 0.44 0.37 Richter (2007) Switzerl. Zofing. 8.2 0–25 >100 517 600 144 0.28 0.24 142 0.27 0.24 Richter (2007) Germany Götting. 8.7 0–15 120 177 219 41 0.23 0.19 75 0.42 0.34 Hertel (1999) Germany Lüneb. 8.1 0–5 100 279 312 458 1.64 1.47 97 0.35 0.31 Hertel (1999) Germany Solling 6.9 0–5 150 134 149 226 1.68 1.51 45 0.33 0.30 Hertel (1999) Germany Ziegel. 8.6 0–10 120 70 100 203 2.92 2.04 46 0.66 0.46 Hertel (1999) Germany Götting. 7.0 0–20 130 195 282 218 1.12 0.77 157 0.81 0.56 Wu (2000) Germany Solling 6.4 0–40 149 328 373 211 0.64 0.57 85 0.26 0.23 Wu (2000) France Aubure 6.0 0–30 161 83 120 165 2.00 1.38 77 0.93 0.64 Stober et al. (2000) Picea abies: Germany Fichtel. 5.3 0–60 140 175 224 304 1.74 1.36 104 0.60 0.47 Gaul et al. (2009) Germany Barbis 8.0 0–40 39 182 235 116 0.63 0.49 124 0.68 0.53 Fritz (1999) Germany Eberg. 7.8 0–40 34 150 188 83 0.56 0.44 90 0.60 0.48 Fritz (1999) Germany Fichtel. 5.5 0–40 40 245 340 156 0.64 0.46 160 0.65 0.47 Fritz (1999) Germany Harz 6.0 0–40 47 204 241 278 1.36 1.15 63 0.31 0.26 Fritz (1999) Estonia Roela 5.4 0–40 60 142 199 251 1.77 1.26 135 0.95 0.68 Ostonen et al. (2005) France Aubure 6.0 0–30 92 57 70 89 1.56 1.27 30 0.52 0.43 Stober et al. (2000) Norway Nordm. Assessment of other factors generating variation in fine-root turnover rates 3.8 0–40 50 462 603 298 0.65 0.49 282 0.61 0.47 Eldhuset et al. (2006) Norway Nordm. 3.8 0–60 60 56 62 63 1.13 1.02 17 0.31 0.27 Børja et al. (2008) Norway Nordm. 3.8 0–60 120 50 63 70 1.40 1.11 22 0.48 0.35 Børja et al. (2008) Sweden Forsm. 5.5 0–40 80 304 410 241 0.79 0.59 186 0.61 0.45 Persson and Stadenberg (2010) ( ) Table 3 Sequential coring: Mean and maximum biomass, annual production, and turnover rate of tree fine roots recorded with sequential coring. The annual production is calculated with the ‘Decision Matrix’ or the ‘Maximum-Minimum’ formula, and the turnover rate is calculated by dividing the annual production by the mean biomass (Bmean) or by the maximum biomass (Bmax). (B 0 Biomass) Country Site Mean annual temp. (°C) Depth (cm) Stand age (yr) Biomass (B) Decision Matrix Maximum-Minimum References Mean (g m−2) Max. (g m−2) Production Turnover rate Production Turnover rate Bmean Bmax Bmean Bmax (g m−2 yr−1) (yr−1) (yr−1) (g m−2 yr−1) (yr−1) (yr−1) 3 Sequential coring: Mean and maximum biomass, annual production, and turnover rate of tree fine roots recorded with sequential coring. The annual production is calculated he ‘Decision Matrix’ or the ‘Maximum-Minimum’ formula, and the turnover rate is calculated by dividing the annual production by the mean biomass (Bmean) or by the mum biomass (Bmax). (B 0 Biomass) ry Site Mean annual temp. (°C) Depth (cm) Stand age (yr) Biomass (B) Decision Matrix Maximum-Minimum References Mean (g m−2) Max. (g m−2) Production Turnover rate Production Turnover rate Bmean Bmax Bmean Bmax (g m−2 yr−1) (yr−1) (yr−1) (g m−2 yr−1) (yr−1) (yr−1) Plant Soil (2013) 362:357–372 364 ab e 3 (co t ued) Country Site Mean annual temp. (°C) Depth (cm) Stand age (yr) Biomass (B) Decision Matrix Maximum-Minimum References Mean (g m−2) Max. (g m−2) Production Turnover rate Production Turnover rate Bmean Bmax Bmean Bmax (g m−2 yr−1) (yr−1) (yr−1) (g m−2 yr−1) (yr−1) (yr−1) Pinus sylvestris: Finland Iloman. 1.9 0–30 38 278 363 862 3.10 2.37 181 0.65 0.50 Makkonen and Helmisaari (1999) Sweden Ivantj. 5.2 0–30 120 120 153 242 2.03 1.58 69 0.58 0.45 Persson (1980a) Populus spp.: Italy P. alba 14.4 0–40 2 110 143 55 0.50 0.39 56 0.51 0.40 Lukac et al. (2003) Italy P. nigra 14.4 0–40 2 109 158 84 0.77 0.53 84 0.77 0.53 Lukac et al. (2003) Italy P. eura. 14.4 0–40 2 146 187 55 0.37 0.29 89 0.61 0.48 Lukac et al. (2003) Quercus ilex/Q. cerrioides: Spain Bages 14.4 0–50 10 858 1336 – – – 812 0.95 0.61 Miguel Pérez (2010) Quercus petraea: France La Croix 8.0 0–55 45 310 346 53 0.17 0.15 63 0.29 0.18 Bakker (1999) Plant Soil (2013) 362:357–372 365 Table 4 Ingrowth cores: Mean and maximum biomass, annual production, and turnover rate of tree fine roots recorded with ingrowth cores. The annual production is calculated with the ‘Decision Matrix’ or the ‘Maximum-Minimum’ formula, and the turnover rate is calculated by dividing the annual production by the mean biomass (Bmean) or by the maximum biomass (Bmax). (B0Biomass) Country Site Mean annual temp. (°C) Depth (cm) Year after install. (yr) Stand age (yr) Biomass (B) Decision Matrix Maximum-Minimum References Mean (g m−2) Max. (g m−2) Production Turnover rate Production Turnover rate Bmean Bmax Bmean Bmax (g m−2 yr−1) (yr−1) (yr−1) (g m−2 yr−1) (yr−1) (yr−1) Fagus sylvatica: Germany Götting. 7.0 0–20 2 130 42 107 107 2.58 1.00 107 2.58 1.00 Wu (2000) Germany Solling 6.4 0–20 2 149 48 123 123 2.57 1.00 123 2.57 1.00 Wu (2000) Picea abies: Switzerl. Schlad. 9.6 0–10 2 70 80 106 65 0.81 0.62 65 0.81 0.62 Genenger et al. (2003) Estonia Roela 5.4 0–30 2 60 52 100 89 1.70 0.89 74 1.41 0.74 Ostonen et al. (2005) Estonia Roela 5.4 0–30 3 60 70 100 66 0.94 0.65 51 0.73 0.51 Ostonen et al. (2005) Pinus sylvestris: Switzerl. Pfynw. 9.2 0–10 2 90 44 62 37 0.84 0.59 37 0.84 0.59 Brunner et al. Soil stratification and root turnover rate double the Maximum-Minimum method. The turnover rates were significantly different when using mean bio- mass data (1.14 yr−1 from the Decision Matrix versus 0.57 yr−1 from the Maximum-Minimum method) as well as when using maximum biomass data (0.88 yr−1 versus 0.43 yr−1). Using mean biomass data resulted in significantly higher turnover rates compared to the use of maximum biomass data (p00.006, Fig. 3), with a mean difference of about 30 %. Our results show that a layer-per-layer approach yields a higher turnover rate than a ‘one soil layer’ approach (Fig. 2). Using average data for the whole of the soil profile, as opposed to using data for individual layers, does not capture all observed differences in root biomass and therefore results in a lower estimate of NPP and thus significantly lower turnover rate T ( Twholeprofile ¼ 0:88 Tlayerperlayer 0:17; r 2 00. 91, p<0.001; Fig. 2). 3 Sequential coring: Mean and maximum biomass, annual production, and turnover rate of tree fine roots recorded with sequential coring. The annual production is calculated he ‘Decision Matrix’ or the ‘Maximum-Minimum’ formula, and the turnover rate is calculated by dividing the annual production by the mean biomass (Bmean) or by the mum biomass (Bmax). (B 0 Biomass) ry Site Mean annual temp. (°C) Depth (cm) Stand age (yr) Biomass (B) Decision Matrix Maximum-Minimum References Mean (g m−2) Max. (g m−2) Production Turnover rate Production Turnover rate Bmean Bmax Bmean Bmax (g m−2 yr−1) (yr−1) (yr−1) (g m−2 yr−1) (yr−1) (yr−1) (2009) Sweden Ivantj. 5.2 – 2 120 65 136 126 1.96 0.93 115 1.78 0.84 Persson (1980a) Plant Soil (2013) 362:357–372 366 Discussion Comparison between the Decision Matrix and the Maximum-Minimum method Decision Matrix versus Maximum-Minimum method Decision Matrix versus Maximum-Minimum method Turnover rates were calculated from the whole data set of sequential coring and using the Decision Matrix and the Maximum-Minimum method 0.0 Mann-Whitney U: Method: p < 0.001 Biomass: p = 0.006 DM DM MM MM a b c Turnover rate (yr-1) Biomassmax 0.4 0.8 1.2 1.6 Biomassmean ab Fig. 3 Mean turnover rates calculated from the whole data set of sequential coring and using the Decision Matrix (DM) or the Maximum-Minimum method (MM) and using mean biomass Bmean or maximum biomass Bmax data y = 1.30 x - 0.001 r2 = 0.98, p < 0.001 Turnover rate Bmean (yr-1) 0 1 2 3 Turnover rate Bmax (yr-1) 0 1 2 3 4 0.0 Mann-Whitney U: Method: p < 0.001 Biomass: p = 0.006 DM DM MM MM a b c Turnover rate (yr-1) Biomassmax 0.4 0.8 1.2 1.6 Biomassmean ab Fig. 3 Mean turnover rates calculated from the whole data set of sequential coring and using the Decision Matrix (DM) or the Maximum-Minimum method (MM) and using mean biomass Bmean or maximum biomass Bmax data Fig. 3 Mean turnover rates calculated from the whole data set of sequential coring and using the Decision Matrix (DM) or the Maximum-Minimum method (MM) and using mean biomass Bmean or maximum biomass Bmax data Fig. 1 Relationship between turnover rates using mean biomass (Bmean) or maximum biomass data (Bmax). Turnover rates were calculated from the whole data set of sequential coring and using the Decision Matrix and the Maximum-Minimum method Fig. 1 Relationship between turnover rates using mean biomass (Bmean) or maximum biomass data (Bmax). Turnover rates were calculated from the whole data set of sequential coring and using the Decision Matrix and the Maximum-Minimum method may cover some ecosystems, it cannot correctly capture reality in systems where faster root turnover has been observed (e.g. Lukac et al. 2003) as it was shown for fast growing species (Jourdan et al. 2008). The Maximum- Minimum method is therefore only suitable for ecosys- tems with strong annual fluctuation of fine root biomass where turnover rate is not expected to exceed 1. In a forest ecosystem where root production and root death occur continuously and on a similar level all year round (e.g. ‘steady state’), no differences between maximum and minimum biomass will be observed. Decision Matrix versus Maximum-Minimum method Mean turnover rates calculated with the Decision Matrix were significantly higher than rates calculated with the Maximum-Minimum method (p<0.001, Fig. 3). The Decision Matrix methods yielded T approximately By analysing our European data set, we found about two times higher root turnover rates when using the Decision Matrix method compared to the Maximum-Minimum method. The observed discrepancy is best described by Table 5 Summary of biomass, annual production, and turnover rates (±SE) of fine roots of common European tree species. The annual production is calculated with the ‘Decision Matrix’ or the ‘Maximum-Minimum’ formula, and the turnover rate is calculated by dividing the annual production by the mean bio- mass (Bmean) or by the maximum biomass (Bmax) Biomass (B) Decision Matrix Maximum-Minimum Mean (g m−2) Maximum (g m−2) Production (g m−2 yr−1) Turnover rate Production (g m−2 yr−1) Turnover rate Bmean (yr−1) Bmax (yr−1) Bmean (yr−1) Bmax (yr−1) Sequential coring method Fagus sylvatica (n013): 327 411 278 1.11 0.86 163 0.53 0.41 (±57) (±71) (±44) (±0.21) (±0.16) (±31) (±0.06) (±0.03) Picea abies (n011): 184 240 177 1.11 0.88 110 0.57 0.44 (±37) (±49) (±30) (±0.14) (±0.11) (±24) (±0.05) (±0.04) Pinus sylvestris (n02): 199 258 552 2.57 1.98 125 0.62 0.48 (±80) (±105) (±310) (±0.54) (±0.40) (±56) (±0.04) (±0.02) Ingrowth cores method Fagus sylvatica (n02): 45 115 115 2.58 1.00 115 2.58 1.00 (±3) (±8) (±8) (±0.01) (±0.00) (±8) (±0.01) (±0.00) Picea abies (n03): 67 102 73 1.15 0.72 63 0.98 0.62 (±8) (±2) (±8) (±0.28) (±0.09) (±7) (±0.21) (±0.07) Pinus sylvestris (n02): 55 99 82 1.40 0.76 76 1.31 0.72 (±11) (±37) (±45) (±0.56) (±0.17) (±39) (±0.47) (±0.12) Plant Soil (2013) 362:357–372 367 y = 1.30 x - 0.001 r2 = 0.98, p < 0.001 Turnover rate Bmean (yr-1) 0 1 2 3 Turnover rate Bmax (yr-1) 0 1 2 3 4 Fig. 1 Relationship between turnover rates using mean biomass (Bmean) or maximum biomass data (Bmax). Turnover rates were calculated from the whole data set of sequential coring and using the Decision Matrix and the Maximum-Minimum method y = 1.30 x - 0.001 r2 = 0.98, p < 0.001 Turnover rate Bmean (yr-1) 0 1 2 3 Turnover rate Bmax (yr-1) 0 1 2 3 4 Fig. 1 Relationship between turnover rates using mean biomass (Bmean) or maximum biomass data (Bmax). Decision Matrix versus Maximum-Minimum method Such an obser- vation will result in a zero estimate of root production and subsequently a zero estimate of root turnover rate (see also Kurz and Kimmins 1987). the fact that Decision Matrix accumulates differences between all observations—the larger the number of interim observations (e.g. monthly observations) the larger the potential for accounting all the peaks and troughs. The Maximum-Minimum method, on the other hand, makes use only of the annual net gain in biomass. On the basis of our comparison, we suggest that the Maximum-Minimum method should be used with cau- tion; by definition, root turnover rates calculated by this method are bound between 0 and 1. Although this range 0 1 2 3 4 0 1 2 3 4 Turnover rate whole profile (yr-1) Turnover rate layer-per-layer (yr-1) y = 0.88 x - 0.17 r2 = 0.91, p< 0.001 Fig. 2 Relationship between turnover rates calculated per whole soils profiles or per individual soil layers (summed versus individual layers). Turnover rates were calculated the whole data set of sequential coring and using the Decision Matrix method and maximum biomass data (data from Hertel 1999; Richter 2007; Makkonen and Helmisaari 1999; Bakker 1999; Ostonen et al. 2005). Mean soil depth is 44 cm, and the average number of individual soil layers is four 0 1 2 3 4 0 1 2 3 4 Turnover rate whole profile (yr-1) Turnover rate layer-per-layer (yr-1) y = 0.88 x - 0.17 r2 = 0.91, p< 0.001 Moving on to the Decision Matrix method, the weak point of this method is—as with all methods using dead roots—the difficulty of quantifying root necromass. The potentially rapid disappearance of root necromass may lead to underestimates (Hendricks et al. 2006). One of possible reasons for the rapid disap- pearance is belowground herbivory (Stevens et al. 2002). Nevertheless, we propose that if necromass observations are available or can be obtained, the Decision Matrix should be favoured over the Minimum-Maximum formula. The former considers both living and dead fine roots, the calculation is thus based on more information, reducing the scope for significant errors. One potential source of error, how- ever, is if all the differences in fine root biomass and necromass between sampling periods are subjected to the calculations regardless of their statistical signifi- cance. This may lead to overestimation. Turnover rate whole profile Turnover rate layer-per-layer (yr-1 Fig. Decision Matrix versus Maximum-Minimum method 2 Relationship between turnover rates calculated per whole soils profiles or per individual soil layers (summed versus individual layers). Turnover rates were calculated the whole data set of sequential coring and using the Decision Matrix method and maximum biomass data (data from Hertel 1999; Richter 2007; Makkonen and Helmisaari 1999; Bakker 1999; Ostonen et al. 2005). Mean soil depth is 44 cm, and the average number of individual soil layers is four Fig. 2 Relationship between turnover rates calculated per whole soils profiles or per individual soil layers (summed versus individual layers). Turnover rates were calculated the whole data set of sequential coring and using the Decision Matrix method and maximum biomass data (data from Hertel 1999; Richter 2007; Makkonen and Helmisaari 1999; Bakker 1999; Ostonen et al. 2005). Mean soil depth is 44 cm, and the average number of individual soil layers is four Plant Soil (2013) 362:357–372 368 Even though root coring methods—whether se- quential or ingrowth—do deliver dependable and comparable measurements of fine root turnover, the application of the minirhizotron technique to estimate fine-root production and turnover is still favoured over the sequential coring or the ingrowth core method in certain situations (Hendricks et al. 2006). Turnover rate estimates obtained by minirhizotron studies can be higher than 1 and the method allows for repeated observation of the same roots. However, in some forest ecosystems, application of minirhizotron meth- ods to measure fine-root production is hampered, e.g. in stony or shallow soils or on steep slopes. Sequential coring and ingrowth core methods are suitable even for these environments, giving them an advantage in terms of comparability of resulting data. between-year fluctuations due to climatic variation, which occur even if a forest ecosystem is at a steady state. Thus, we propose that mean biomass rather than the maximum is more representative of the annual live biomass present in the soil. The use of mean biomass in our calculations increased the turnover rates by about 30 % compared to the use of the maximum biomass. An additional factor significantly affecting the results of the turnover calculations is the use of summed up values of biomass, necromass, and pro- ductivity for the whole soil profile versus using these data for individual soil layers (horizons). Decision Matrix versus Maximum-Minimum method We acknowl- edge that using individual horizons should be prefer- able as the root turnover rate may be affected by differing physical and chemical characteristics of in- dividual horizons. We established that basing root turnover rate calculation on individual horizon data increases the overall turnover rate—probably because it allows for better capture of biomass and necromass variations over time. We are, however, aware that root biomass and production observation on a horizon basis constitutes a significant technical challenge and contend that using whole-soil data is acceptable. Further factors potentially influencing the turnover rate, e.g. soil depth, length of study, or root diameter class have also been tested in this study, however, the available European dataset for these parameters was limited and did not allow further deductions. Thus, besides the uncertainties due to climatic and calculation reasons, many other external factors may potentially affect the estimates of root turnover rates. At present, no available technique can solve this predicament and we put forward that our root turnover rates represent the best approximation obtained by using sequential soil or ingrowth cores. Maximum biomass versus mean biomass By definition, the denominator in the root turnover calculation equation is the representation of biomass present in the soil. An assumption inherent to all root turnover calculation method is that annual fine root production (obtained by whatever method) equals to fine root mortality and the system is at steady state on an annual basis. Over the course of a year, new growth replaces roots which have died. The proportion of roots which have been replaced can therefore be cal- culated as root production over biomass. At the pres- ent, both maximum and mean root biomass are used, with about two-thirds of studies using maximum bio- mass (Gill and Jackson 2000). They justified the use of the maximum biomass as “…because it is an ex- tensively used model of root turnover and because of its heuristic value”. When constructing models of root allocation in forests, a case can be made for maximum biomass to be the preferred parameter over mean or minimum values due to the importance of setting an upper limit for the allocation rate. Fine root allocation rate may depend on sink strength (C demand), but might ultimately be limited by the maximum fraction of GPP which trees can allocate to root systems (Astrid Meyer, personal communication; see also Farrar and Jones 2000; Gower et al. 1996; Poorter et al. 2012). Having said that, and bearing in mind that the root turnover calculation assumes an ecosystem at steady state, a mean value is indicative of the long-term average as it evens out seasonal variation in biomass. Maximum biomass, on the other hand, is substantially more susceptible to Turnover rates of European tree species Turnover rates of European tree species Our review of published studies from European forest stands revealed that most data for fine-root turnover rate originate from sequential coring, with the preva- lence of Fagus sylvatica or Picea abies as the species of interest. Studies performed in forest stands with other dominating tree species such as Quercus spp., Pinus spp. were far less abundant. Similarly, turnover rate studies where ingrowth cores were used instead of employing the sequential coring method to measure fine-root production, were far less abundant. Whereas in our study the data sets of F. sylvatica derived mainly from Central Europe, the data sets of P. abies originated Plant Soil (2013) 362:357–372 369 from Central as well as from Northern Europe. Trees from Southern European countries were represented only by a few data sets, and no conclusive turnover rates can be suggested for this environment yet. Overall, we propose that only the fine root turnover rates in our study for the following species may be recommended for further use in biogeochemical models with a reason- able degree of accuracy: F. sylvatica and P. abies. We established a turnover rate of 1.11 yr−1 for both F. sylvatica and P. abies, using the Decision Matrix for- mula and the mean biomass data from sequential coring. root turnover rates are used, some resembling measured values, others less so. In one of the first applications, the fine-root turnover rate was set to 1.0 yr−1 for deciduous broad-leaf and deciduous needle-leaf trees and to 0.26 yr−1 for evergreen needle-leaf trees (White et al. 2000, using the Biome-BGC model). The distinct differ- ence between deciduous trees and evergreen needle-leaf trees mainly originated from the notion that fine-root turnover rate is equal to leaf turnover rate. A compilation of the various turnover rates applied in European model- ling studies is shown in Table 6. Most recent studies applied a universal fine-root turnover rate of 0.7 yr−1 to all forest tree species (Hickler et al. 2008, using the LPJ- GUESS model). This assumption is based on Vogt et al. (1996) and on Li et al. (2003) (Thomas Hickler, personal communication). Li et al. (2003) found a linear relation- ship between fine root production and fine root biomass, with the turnover rate 0.64 yr−1 which was lower than the original estimate of 0.73 y−1 from a previous analysis (Kurz et al. 1996). a and Tatarinov and Cienciala (2006) Turnover rates of European tree species Using ‘universal’ turnover rates, how- ever, should be discouraged if country-based C budgets have to be reported within the frame to the Kyoto Conclusions Eldhuset TD, Lange H, de Wit HA (2006) Fine root biomass, necromass and chemistry during seven years of elevated aluminium concentrations in the soil solution of a middle- aged Picea abies stand. Sci Total Environ 369:344–356 The present synthesis on fine-root turnover of European forests reveals that only Fagus sylvatica and Picea abies have sufficient data availability to suggest mean turnover rates obtained by soil coring to be used by National C reporters (0.86±0.16 yr−1 for F. sylvatica, 0.88±0.11 yr−1 for P. abies, when maximum biomass data are used; 1.11±0.21 yr−1 for F. sylvatica, 1.11± 0.14 yr−1 for P. abies, when mean biomass data are used). Data sets of other European forests or obtained by alternative methods such as ingrowth cores were too small to allow for distinct conclusions on the turnover rates. Based on our calculations, we put forward that usage of mean rather than maximum root biomass in turnover calculations is preferable as it better reflects long-term quantity of biomass. aged Picea abies stand. Sci Total Environ 369:344–3 Endrulat T, Saurer M, Buchmann N, Brunner I (2010) Incorpo- ration and remobilization of 13C within the fine-root systems of individual Abies alba trees in a temperate coniferous stand. Tree Physiol 30:1515–1527 Fairley RI, Alexander IJ (1985) Methods of calculating fine root production in forests. In: Fitter AH, Atkinson D, Read DJ (eds) Ecological interactions in soil: plants, microbes and animals. Blackwell, Oxford, pp 37–42 Farrar JF, Jones DL (2000) The control of carbon acquisition by roots. New Phytol 147:43–53 Finer L, Ohashi M, Noguchi K, Hirano Y (2011) Fine root production and turnover in forest ecosystems in relation to stand and environmental characteristics. For Ecol Manag 262:2008–2023 Fritz HW (1999) Feinwurzel-Verteilung, -Vitalität, -Produktion und -Umsatz von Fichten (Picea abies (L.) Karst.) auf unterschiedlich versauerten Standorten. Ber Forsch Zent Waldökosyst, Reihe A 165:1–138 Gaudinski JB, Trumbore SE, Davidson EA, Cook AC, Makrewitz D, Richter DD (2001) The age of fine-root carbon in three forests of the eastern United States measured by radiocarbon. Oecologia 129:420–429 Acknowledgements This study is a result of the activity of a Working Group of the COSTAction FP0803 “Belowground carbon turnover in European forests”. We thank Astrid Meyer and Thomas Hickler for helpful comments on the calculations and the models, and Beat Frey for critical reading. Turnover rates applied in biogeochemical models Turnover rates applied in biogeochemical models One of the aims of the present study was to deliver suitable fine-root turnover data of European tree species, which may be used by modellers to construct ecosystem or biogeochemical models. Such models are applied in many European countries to report the change of below- ground C in European forests as a reporting requirement for the Kyoto protocol signatories. A brief overview of the models applied so far shows that a wide variety of Table 6 Fine-root turnover rates (yr−1) of European trees used in biogeochemical models. (BGC 0 Biogeochemical cycles, GUESS 0 General ecosystem simulator, LPJ 0 Lund-Potsdam-Jena model) Tree type Tree species Turnover rate Model Reference Broad-/Deciduous needle-leaved 1.0 Biome-BGC White et al. (2000) Broad-leaved summergreen 1.0 LPJ-GUESS Smith et al. (2001) Broad-leaved 1.0 LPJ-GUESS Hickler et al. (2004) Broad-leaved 0.7 LPJ-GUESS Hickler et al. (2006, 2008) Fagus sylvatica 1.023 Biome-BGC Cienciala and Tatarinov (2006)a Fagus sylvatica 1.0 Biome-BGC Pietsch et al. (2005) Quercus robur 1.023 Biome-BGC Cienciala and Tatarinov (2006)a Quercus robur 1.0 Biome-BGC Pietsch et al. (2005) Quercus petraea 1.023 Biome-BGC Cienciala and Tatarinov (2006)a Quercus petraea 1.0 Biome-BGC Pietsch et al. (2005) Larix decidua 1.0 Biome-BGC Pietsch et al. (2005) Evergreen needle-leaved 0.26 Biome-BGC White et al. (2000) Needle-/Broad-leaved evergreen 0.5 LPJ-GUESS Smith et al. (2001) Needle-leaved 0.5 LPJ-GUESS Hickler et al. (2004) Needle-leaved 0.7 LPJ-GUESS Hickler et al. (2006, 2008) Picea abies 0.811 Biome-BGC Cienciala and Tatarinov (2006)a Picea abies 0.195 Biome-BGC Pietsch et al. (2005) Pinus sylvestris 0.18 Biome-BGC Pietsch et al. (2005) Pinus cembra 0.18 Biome-BGC Pietsch et al. (2005) a and Tatarinov and Cienciala (2006) ates (yr−1) of European trees used in biogeochemical models. (BGC 0 Biogeochemical cycles, GUESS 0 , LPJ 0 Lund-Potsdam-Jena model) 370 Plant Soil (2013) 362:357–372 supply in a dry climatic region in Switzerland. Tree Physiol 29:541–550 protocol and species-specific and biome based values of root turnover rate are available. Cienciala E, Tatarinov FA (2006) Application of BIOME-BGC model to managed forests. 2. Comparison with long-term observations of stand production for major tree species. For Ecol Manag 237:252–266 Conclusions We also would like to thank Carmen Biel, Karna Hansson, Peter Zeleznik, Thomas Leski, Inger- Kappel Schmidt, Antonino di Iorio, Kyotaro Noguchi, and Yoni Ephrat, who helped us in any way to write up this review. Claude Herzog helped with statistical tests. Gaudinski JB, Tom MS, Riley WJ, Dawson TE, Joslin JD, Majdi H (2010) Measuring and modeling the spectrum of fine-root turnover times in three forests using isotopes, minirhizotrons, and the Radix model. Glob Biogeochem Cycles 24:GB3029 Gaul D, Hertel D, Leuschner C (2009) Estimating fine root longevity in a temperate Norway spruce forest using three independent methods. Funct Plant Biol 36:11–19 Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. 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https://openalex.org/W2917138662	https://europepmc.org/articles/pmc6406696?pdf=render	English	null	Integrative Omic Profiling Reveals Unique Hypoxia Induced Signatures in Gastric Cancer Associated Myofibroblasts	Cancers	2,019	cc-by	11,815	Integrative Omic Proﬁling Reveals Unique Hypoxia Induced Signatures in Gastric Cancer Associated Myoﬁbroblasts Hanna Najgebauer 1,2,* , Andrew F. Jarnuczak 2 , Andrea Varro 1 and Christopher M. Sanderson 1 1 Department of Cellular and Molecular Physiology, University of Liverpool, Crown Street, Liverpool L69 3BX, UK; A.Varro@liverpool.ac.uk (A.V.); C.Sanderson@liverpool.ac.uk (C.M.S.) 2 European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK; jarnuczak@ebi.ac.uk * Correspondence: hnajgebauer@ebi.ac.uk or hanna.najgebauer@gmail.com 1 Department of Cellular and Molecular Physiology, University of Liverpool, Crown Street, Liverpool L69 3BX, UK; A.Varro@liverpool.ac.uk (A.V.); C.Sanderson@liverpool.ac.uk (C.M.S.) 2 European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK; jarnuczak@ebi.ac.uk * Correspondence: hnajgebauer@ebi.ac.uk or hanna.najgebauer@gmail.com Received: 30 January 2019; Accepted: 20 February 2019; Published: 23 February 2019 Abstract: Although hypoxia is known to contribute to several aspects of tumour progression, relatively little is known about the effects of hypoxia on cancer-associated myoﬁbroblasts (CAMs), or the consequences that conditional changes in CAM function may have on tumour development and metastasis. To investigate this issue in the context of gastric cancer, a comparative multiomic analysis was performed on populations of patient-derived myoﬁbroblasts, cultured under normoxic or hypoxic conditions. Data from this study reveal a novel set of CAM-speciﬁc hypoxia-induced changes in gene expression and secreted proteins. Signiﬁcantly, these signatures are not observed in either patient matched adjacent tissue myoﬁbroblasts (ATMs) or non-cancer associated normal tissue myoﬁbroblasts (NTMs). Functional characterisation of different myoﬁbroblast populations shows that hypoxia-induced changes in gene expression not only enhance the ability of CAMs to induce cancer cell migration, but also confer pro-tumorigenic (CAM-like) properties in NTMs. This study provides the ﬁrst global mechanistic insight into the molecular changes that contribute to hypoxia-induced pro-tumorigenic changes in gastric stromal myoﬁbroblasts. Keywords: cancer-associated myoﬁbroblasts; hypoxia; tumour microenvironment; gene expression; secretome; transcriptomics; proteomics; gastric cancer; omics cancers cancers cancers cancers cancers 1. Introduction Gastric cancer remains a leading cause of cancer death worldwide [1]. It is now clear that many tumours develop as a result of complex reciprocal interactions between cancer cells and neighbouring cells within the tumour microenvironment. These include ﬁbroblasts, myoﬁbroblasts, endothelial cells, and immune cells, which together contribute to the formation of a specialised extracellular matrix, and soluble paracrine factors [2,3] which support the growth and metastasis of gastric tumours. In contrast to many other tissues, ‘activated’ myoﬁbroblasts are well represented in normal gastric tissue, where they are closely allied to the epithelium. However, in gastric cancers, resident myoﬁbroblasts acquire additional tumour promoting functions [4]. In particular, gastric cancer-associated myoﬁbroblasts (CAMs) secrete factors that increase the migration, invasion, and proliferation of cancer cells, when compared to either adjacent tissue myoﬁbroblasts (ATMs), or normal tissue myoﬁbroblasts (NTMs) [5]. This tumour-promoting phenotype is maintained, at least in part, due to a robust CAM-speciﬁc DNA methylation signature [6]. Analysis of gastric CAM conditioned media conﬁrmed that these cells exhibit distinct patterns of protein secretion, Cancers 2019, 11, 263; doi:10.3390/cancers11020263 www.mdpi.com/journal/cancers Cancers 2019, 11, 263 2 of 18 compared to tissue matched ATMs. Further, a direct correlation was observed between decreased levels of extracellular matrix adaptor proteins, such as transforming growth factor β-induced gene-h3 (TGFβig-h3) in the secretome of gastric CAMs and the occurrence of lymph node metastasis, worse prognosis, and shorter patient survival times [5]. Signiﬁcantly, a number of other factors that distinguish gastric CAMs from normal non-cancer associated myoﬁbroblasts have been reported. One such factor is galectin-1, which shows increased expression in CAMs and was shown to enhance gastric cancer cell migration and invasion by upregulating integrin-β1 expression [7]. In addition, CAM-derived galectin-1 is also known to promote angiogenesis in gastric cancer [8]. Other examples of CAM speciﬁc changes include the upregulation of miR-106b and ﬁbroblast growth factor 9 (FGF-9) [9,10]. FGF-9 promotes the anti-apoptotic and invasive capability of gastric cancer cells [9], whereas upregulated miR-106b is associated with poor patient prognosis, as it enhances gastric cancer cell migration and invasion [10]. Immunohistochemistry and real-time PCR experiments have shown that CAMs frequently accumulate in the stroma of gastric tumours, and the prevalence of CAMs shows a positive correlation with tumour size, depth, and metastasis [11]. 1. Introduction Interestingly, studies in scirrhous gastric cancer, which has the worst prognosis among all types of gastric cancer, show that CAMs may also regulate the stemness of cancer stem cells (CSCs) [12], identifying Asporin as a unique CAM-derived secretory protein that promotes the coordinated invasion of both CAMs and cancer cells [13]. As CAMs play a vital role in the progression of gastric cancer, it is critical to deﬁne the molecular mechanisms that enable gastric CAMs to facilitate different aspects of tumour progression. This study was designed to compare the molecular modulation of different myoﬁbroblasts populations (CAM, ATM, NTM) in response to hypoxic conditions. Hypoxia is an important microenvironmental factor which contributes to several aspects of tumour progression, including tumour growth, tissue invasion, and metastatic spread [14,15]. Although signiﬁcant efforts have been devoted to studying the consequences of hypoxia on cancer cells, relatively little is known about the effects or consequence of hypoxia on the stromal component of the tumour microenvironment. Previously, it was proposed that hypoxia may have a role in modulating the differentiation and activity of stromal cells [16] in such a way that they synergise with hypoxic oxidative stress to enhance tumour aggressiveness in melanomas [17]. In this context, hypoxia-mediated oxidative stress was found to be mandatory for the activation of dermal ﬁbroblasts and secretion of cytokines and other pro-migratory factors, in addition to actively promoting the invasion and chemotaxis of melanoma cells [17]. However, the extent to which exposure to hypoxia may contribute to or change myoﬁbroblast activity in gastric cancer remains largely unexplored. For example, it is not yet known if hypoxia induces a more aggressive CAM phenotype, or if NTMs become more aggressive, or more CAM-like, under hypoxic conditions. Equally, little is known about the induced conditional changes in gene expression that may facilitate pro-tumorigenic changes in myoﬁbroblasts, including changes in the secretome of CAMs or NTMs, which in turn may have the potential to impose changes in cancer cell function. To address these questions, we employed a combination of cell-based assays and genomic, transcriptomic, and proteomics approaches. Results from this study show that hypoxia enhances CAM-induced cancer cell migration and promotes the acquisition of CAM-like properties in NTMs. 2. Results 2.1. Hypoxia Enhances CAM-Induced Cancer Cell Migration and Promotes CAM-Like Properties in NTM To assess how hypoxia (1% O2) may affect the ability of CAMs and NTMs to induce cancer cell migration and/or proliferation, a series of complementary Boyden chamber migration and EdU cell proliferation assays were performed, in which gastric adenocarcinoma cells (AGS) were exposed to fresh CAM or NTM conditioned media (CM), prepared under hypoxic (hypoxic-CM) or normoxic (ctrl-CM) conditions. Results from these studies show that both CAM- and NTM-derived hypoxic-CM signiﬁcantly enhanced the migration of gastric cancer cells, in comparison to corresponding ctrl-CM derived from either CAMs or NTMs (Figure 1A, Figure S1A). These data conﬁrm that hypoxia induces the expression and release of pro-migratory factors. Interestingly, group mean data show this effect is on average almost twice as large for CAM-hypoxic-CM, as compared to NTM-hypoxic-CM (Figure 1B). By contrast, hypoxia exerted a reciprocal trend with respect to gastric cancer cell proliferation. Compared to ctrl-CM, CAM-hypoxic-CM consistently reduced gastric cancer cell proliferation, while NTM-hypoxic-CM induced a reproducible increase in cancer cell proliferation (Figure 1C, Figure S1B). On average, exposure to hypoxia conferred a ~40% decrease in CAM-stimulated cancer cell proliferation, in contrast to a ~30% hypoxia-induced increase in NTM-stimulated cancer cell proliferation (Figure 1D). Together, these results suggest that hypoxic conditions may promote some CAM-like properties in gastric NTMs. In particular, exposure to hypoxic conditions would allow non-cancer conditioned NTMs within the surrounding stroma to contribute to hypoxia-enhanced proliferation and spread of metastatic gastric cancer cells. To investigate the molecular mechanisms that confer hypoxia-induced changes in different myoﬁbroblast populations, a comparative global gene expression analysis was performed on patient-matched gastric CAMs and ATMs, and unrelated NTMs. 2.2. Hypoxia Confers Differential Changes in Gene Expression in Gastric Myoﬁbroblasts Puriﬁed from Different Tissue Microenvironments To compare hypoxia-induced gene expression signatures in gastric myoﬁbroblasts puriﬁed from different tissue microenvironments, Illumina HumanHT-12v4 Expression BeadChip arrays were performed on a collection of patient-matched CAM and ATM samples and unrelated NTM samples. In each case, myoﬁbroblasts were cultured in parallel under either hypoxic (1% O2) or normoxic (21% O2) conditions for 72 h. Overall, 13381 genes were consistently expressed in all samples. Of those, 2467 genes were found to be differentially expressed in hypoxic CAMs compared to control CAMs (Figure 2A). In comparison, 2722 genes were found to be signiﬁcantly altered in hypoxic vs. 1. Introduction Furthermore, our data identify unique gene expression and protein secretion signatures between myoﬁbroblasts puriﬁed from different tissue microenvironments, thereby providing a new resource to guide mechanistic investigation into the molecular processes that drive key aspects of tumour stromal communication. Cancers 2019, 11, 263 3 of 18 2. Results normoxic NTMs (Figure 2B), and 2561 genes were differentially expressed in hypoxic vs. normoxic ATMs (Figure 2C). Notably, unsupervised hierarchical clustering correctly grouped samples by condition and identiﬁed clear clusters of genes, which reﬂect transcriptome remodelling in response to hypoxia (Figure 2). 2.2.1. Differential Hypoxia-Induced Myoﬁbroblast Phenotypes Are Not Due to Changes in DNA Methylation To assess whether observed changes in hypoxia-induced gene expression proﬁles are the result of conditional changes in DNA methylation, parallel Illumina Inﬁnium HumanMethylation450 BeadChip assays were performed on CAMs, patient-matched ATMs, and unrelated NTMs. Comparison of genome–wide DNA methylation proﬁles between: (i) CAM hypoxia vs. CAM normoxia, (ii) ATM hypoxia vs. ATM normoxia, and (iii) NTM hypoxia vs. NTM normoxia showed a striking similarity in DNA methylation proﬁles between matched hypoxic and normoxic samples. In all three comparisons, no signiﬁcant methylation changes were observed. For clarity, this is visualised in a single volcano plot and scatter plot in Figure 3A,B. These data suggest that under the conditions used in this study, hypoxia-imposed changes in gene expression are not conferred by associated changes in DNA methylation patterns. 4 of 18 patient- Cancers 2019, 11, 263 myofibroblast pop Figure 1. Differential effects of cancer-associated myofibroblasts (CAM) and normal tissue myofibroblasts (NTM) hypoxic conditioned media (CM) on gastric adenocarcinoma cell (AGS) migration and proliferation. (A,B). CAM-hypoxic-CM (magenta) and NTM-hypoxic-CM (blue) induce AGS cell migration compared to respective CAM or NTM normoxic-control CM (grey). (A) Individual patient data corrected for AGS basal migration (serum-free media) normalised to Figure 1. Differential effects of cancer-associated myoﬁbroblasts (CAM) and normal tissue myoﬁbroblasts (NTM) hypoxic conditioned media (CM) on gastric adenocarcinoma cell (AGS) migration and proliferation. (A,B). CAM-hypoxic-CM (magenta) and NTM-hypoxic-CM (blue) induce AGS cell migration compared to respective CAM or NTM normoxic-control CM (grey). (A) Individual patient data corrected for AGS basal migration (serum-free media) normalised to respective patient-speciﬁc control CM obtained from normoxia (expressed as 100% to enable comparison between effects of CAM and NTM hypoxic conditioned media on AGS migration; for pair-wise comparisons between respective patient-speciﬁc ctrl- and hypoxic-CM, see Supplementary Figure S1A); paired t-test p-value < 0.0001. Error bars represent SD of technical replicates. (B) Group mean data (biological replicates) of AGS cell migration in response to hypoxic-CM from CAMs (n = 3) and NTMs (n = 3); unpaired t-test p-value > 0.05; Error bars represent SEM. (C,D). NTM-hypoxic-CM (blue) induces AGS cell proliferation, whereas CAM-hypoxic-CM (magenta) reduces the ability of AGS cells to proliferate compared to respective CAM or NTM control CM (grey) obtained from Figure 1. Differential effects of cancer-associated myofibroblasts (CAM) and normal tissue myofibroblasts (NTM) hypoxic conditioned media (CM) on gastric adenocarcinoma cell (AGS) migration and proliferation. (A,B). 2.2.1. Differential Hypoxia-Induced Myoﬁbroblast Phenotypes Are Not Due to Changes in DNA Methylation CAM-hypoxic-CM (magenta) and NTM-hypoxic-CM (blue) induce AGS cell migration compared to respective CAM or NTM normoxic-control CM (grey). (A) Individual patient data corrected for AGS basal migration (serum-free media) normalised to Figure 1. Differential effects of cancer-associated myoﬁbroblasts (CAM) and normal tissue myoﬁbroblasts (NTM) hypoxic conditioned media (CM) on gastric adenocarcinoma cell (AGS) migration and proliferation. (A,B). CAM-hypoxic-CM (magenta) and NTM-hypoxic-CM (blue) induce AGS cell migration compared to respective CAM or NTM normoxic-control CM (grey). (A) Individual patient data corrected for AGS basal migration (serum-free media) normalised to respective patient-speciﬁc control CM obtained from normoxia (expressed as 100% to enable comparison between effects of CAM and NTM hypoxic conditioned media on AGS migration; for pair-wise comparisons between respective patient-speciﬁc ctrl- and hypoxic-CM, see Supplementary Figure S1A); paired t-test p-value < 0.0001. Error bars represent SD of technical replicates. (B) Group mean data (biological replicates) of AGS cell migration in response to hypoxic-CM from CAMs (n = 3) and NTMs (n = 3); unpaired t-test p-value > 0.05; Error bars represent SEM. (C,D). NTM-hypoxic-CM (blue) induces AGS cell proliferation, whereas CAM-hypoxic-CM (magenta) reduces the ability of AGS cells to proliferate compared to respective CAM or NTM control CM (grey) obtained from normoxia. (C) Individual patient data corrected for AGS basal proliferation (serum-free media) and normalised to respective patient-speciﬁc control CM obtained from normoxia (expressed as 100% to enable comparison between effects of CAM and NTM hypoxic conditioned media on AGS proliferation; for pair-wise comparisons between respective patient-speciﬁc ctrl- and hypoxic-CM, see Figure S1B); paired t-test p-value < 0.005. Error bars represent SEM of technical replicates. (D) Group mean data (biological replicates) of AGS cell proliferation in response to hypoxic-CM from CAMs (n = 3) and NTMs (n = 3); unpaired t-test p-value < 0.005; Error bars represent SEM. 5 of 18 ypoxia Cancers 2019, 11, 263 g and identified cle Cancers 2019, 11, 263 5 and identified clear clusters of genes, which reflect transcriptome remodelling in response to hypo (Figure 2). Cancers 2018, 10, x 5 o Figure 2. Hypoxia-induced gene expression signatures in gastric myofibroblasts purified from different tissue microenvironments. (A) CAM hypoxia vs. CAM normoxia. (B) NTM hypoxia vs. NTM normoxia. (C) ATM hypoxia vs. ATM normoxia. Left: volcano plots show the distribution of log2 fold changes (x-axis) versus -log10 p-values (y-axis) in respective comparisons. Significantly changing genes (FDR (False discovery rate) p-value < 0.05) are highlighted in black. 2.2.1. Differential Hypoxia-Induced Myoﬁbroblast Phenotypes Are Not Due to Changes in DNA Methylation (A) Volcano plot showing significance versus Δβ values for 424383 methylation probes in the three hypoxia (CAM, ATM, and NTM; n = 9) vs. normoxia (CAM, ATM and NTM; n = 9) comparisons. (B) Scatter plot representing mean β values for normoxic (CAM, ATM and NTM; n = 9) and hypoxic (CAM, ATM, and NTM; n = 9) myofibroblasts. Red dashed lines represent \|Δβ\| > 0.2. C,D. Comparison of hypoxia-induced gene expression signatures identified in gastric CAMs, ATMs, and NTMs. (C) Venn diagram showing the overlap between significantly changed genes (limma p-value < 0.05) in response to hypoxia in CAM, Figure 3. Hypoxia-induced gene expression signatures are not conferred by associated changes in DNA methylation. (A,B) Comparative global DNA methylation proﬁles of primary gastric myoﬁbroblasts following exposure to hypoxic or normoxic conditions. (A) Volcano plot showing signiﬁcance versus ∆β values for 424383 methylation probes in the three hypoxia (CAM, ATM, and NTM; n = 9) vs. normoxia (CAM, ATM and NTM; n = 9) comparisons. (B) Scatter plot representing mean β values for normoxic (CAM, ATM and NTM; n = 9) and hypoxic (CAM, ATM, and NTM; n = 9) myoﬁbroblasts. Red dashed lines represent \|∆β\| > 0.2. (C,D). Comparison of hypoxia-induced gene expression signatures identiﬁed in gastric CAMs, ATMs, and NTMs. (C) Venn diagram showing the overlap b t i iﬁ tl h d (li l 0 05) i t h i i CAM ATM Figure 3. Hypoxia-induced gene expression signatures are not conferred by associated changes in DNA methylation. (A,B) Comparative global DNA methylation profiles of primary gastric myofibroblasts following exposure to hypoxic or normoxic conditions. (A) Volcano plot showing significance versus Δβ values for 424383 methylation probes in the three hypoxia (CAM, ATM, and NTM; n = 9) vs. normoxia (CAM, ATM and NTM; n = 9) comparisons. (B) Scatter plot representing mean β values for normoxic (CAM, ATM and NTM; n = 9) and hypoxic (CAM, ATM, and NTM; n = 9) myofibroblasts. Red dashed lines represent \|Δβ\| > 0.2. C,D. Comparison of hypoxia-induced gene expression signatures identified in gastric CAMs, ATMs, and NTMs. (C) Venn diagram showing the overlap between significantly changed genes (limma p-value < 0.05) in response to hypoxia in CAM, ATM, and NTM cells. The common intersection represents a universal hypoxia-induced gene signature. Unique CAM-, ATM-, and NTM- hypoxia-induced gene signatures are highlighted in bold. 2.2.1. Differential Hypoxia-Induced Myoﬁbroblast Phenotypes Are Not Due to Changes in DNA Methylation (A) Volcano plot showing signiﬁcance ver ∆β values for 424383 methylation probes in the three hypoxia (CAM, ATM, and NTM; n = 9) normoxia (CAM, ATM and NTM; n = 9) comparisons. (B) Scatter plot representing mean β values normoxic (CAM, ATM and NTM; n = 9) and hypoxic (CAM, ATM, and NTM; n = 9) myoﬁbrobla Red dashed lines represent \|∆β\| > 0.2. (C,D). Comparison of hypoxia-induced gene express signatures identiﬁed in gastric CAMs, ATMs, and NTMs. (C) Venn diagram showing the over between signiﬁcantly changed genes (limma p-value < 0.05) in response to hypoxia in CAM, AT and NTM cells. The common intersection represents a universal hypoxia-induced gene signatu Figure 3. Hypoxia-induced gene expression signatures are not conferred by associated changes in DNA methylation. (A,B) Comparative global DNA methylation profiles of primary gastric myofibroblasts following exposure to hypoxic or normoxic conditions. (A) Volcano plot showing significance versus Δβ values for 424383 methylation probes in the three hypoxia (CAM, ATM, and NTM; n = 9) vs. normoxia (CAM, ATM and NTM; n = 9) comparisons. (B) Scatter plot representing mean β values for normoxic (CAM, ATM and NTM; n = 9) and hypoxic (CAM, ATM, and NTM; n = 9) myofibroblasts. Red dashed lines represent \|Δβ\| > 0.2. C,D. Comparison of hypoxia-induced gene expression signatures identified in gastric CAMs, ATMs, and NTMs. (C) Venn diagram showing the overlap between significantly changed genes (limma p-value < 0.05) in response to hypoxia in CAM, ATM, and NTM cells. The common intersection represents a universal hypoxia-induced gene signature. Unique CAM-, ATM-, and NTM- hypoxia-induced gene signatures are highlighted in bold. (D) Genes universally changed in the same direction under hypoxia across all three populations of gastric myofibroblasts (CAMs, ATMs, and NTMs, limma FDR p-value < 0.05). Figure 3. Hypoxia-induced gene expression signatures are not conferred by associated changes in DNA methylation. (A,B) Comparative global DNA methylation proﬁles of primary gastric myoﬁbroblasts following exposure to hypoxic or normoxic conditions. (A) Volcano plot showing signiﬁcance versus ∆β values for 424383 methylation probes in the three hypoxia (CAM, ATM, and NTM; n = 9) vs. normoxia (CAM, ATM and NTM; n = 9) comparisons. (B) Scatter plot representing mean β values for normoxic (CAM, ATM and NTM; n = 9) and hypoxic (CAM, ATM, and NTM; n = 9) myoﬁbroblasts. Red dashed lines represent \|∆β\| > 0.2. (C,D). 2.2.1. Differential Hypoxia-Induced Myoﬁbroblast Phenotypes Are Not Due to Changes in DNA Methylation Dashed lines correspond to a 1.6-fold change (log2 fold change of 0.58). Points >0 signify upregulation by hypoxia. Right: Unsupervised hierarchical clustering of the differentially expressed genes (shown in black in volcano plots). Figure 2. Hypoxia-induced gene expression signatures in gastric myoﬁbroblasts puriﬁed from different tissue microenvironments. (A) CAM hypoxia vs. CAM normoxia. (B) NTM hypoxia vs. NTM normoxia. (C) ATM hypoxia vs. ATM normoxia. Left: volcano plots show the distribution of log2 fold changes (x-axis) versus -log10 p-values (y-axis) in respective comparisons. Signiﬁcantly changing genes (FDR (False discovery rate) p-value < 0.05) are highlighted in black. Dashed lines correspond to a 1.6-fold change (log2 fold change of 0.58). Points >0 signify upregulation by hypoxia. Right: Unsupervised hierarchical clustering of the differentially expressed genes (shown in black in volcano plots). 5 o cers 2018, 10, x Figure 2. Hypoxia-induced gene expression signatures in gastric myofibroblasts purified from different tissue microenvironments. (A) CAM hypoxia vs. CAM normoxia. (B) NTM hypoxia vs. NTM normoxia. (C) ATM hypoxia vs. ATM normoxia. Left: volcano plots show the distribution of log2 fold changes (x-axis) versus -log10 p-values (y-axis) in respective comparisons. Significantly changing genes (FDR (False discovery rate) p-value < 0.05) are highlighted in black. Dashed lines correspond to a 1.6-fold change (log2 fold change of 0.58). Points >0 signify upregulation by hypoxia. Right: Unsupervised hierarchical clustering of the differentially expressed genes (shown in black in volcano l t ) Figure 2. Hypoxia-induced gene expression signatures in gastric myoﬁbroblasts puriﬁed from different tissue microenvironments. (A) CAM hypoxia vs. CAM normoxia. (B) NTM hypoxia vs. NTM normoxia. (C) ATM hypoxia vs. ATM normoxia. Left: volcano plots show the distribution of log2 fold changes (x-axis) versus -log10 p-values (y-axis) in respective comparisons. Signiﬁcantly changing genes (FDR (False discovery rate) p-value < 0.05) are highlighted in black. Dashed lines correspond to a 1.6-fold change (log2 fold change of 0.58). Points >0 signify upregulation by hypoxia. Right: Unsupervised hierarchical clustering of the differentially expressed genes (shown in black in volcano plots). 6 of 18 Cancers 2019, 11, 263 Cancers 2018, 10, x 6 of 18 Figure 3. Hypoxia-induced gene expression signatures are not conferred by associated changes in DNA methylation. (A,B) Comparative global DNA methylation profiles of primary gastric myofibroblasts following exposure to hypoxic or normoxic conditions. 2.2.1. Differential Hypoxia-Induced Myoﬁbroblast Phenotypes Are Not Due to Changes in DNA Methylation (D) Genes universally changed in the same direction under hypoxia across all three populations of gastric myofibroblasts (CAMs, ATMs, and NTMs, limma FDR p-value < 0.05). Figure 3. Hypoxia-induced gene expression signatures are not conferred by associated changes in DNA methylation. (A,B) Comparative global DNA methylation proﬁles of primary gastric myoﬁbroblasts following exposure to hypoxic or normoxic conditions. (A) Volcano plot showing signiﬁcance versus ∆β values for 424383 methylation probes in the three hypoxia (CAM, ATM, and NTM; n = 9) vs. normoxia (CAM, ATM and NTM; n = 9) comparisons. (B) Scatter plot representing mean β values for normoxic (CAM, ATM and NTM; n = 9) and hypoxic (CAM, ATM, and NTM; n = 9) myoﬁbroblasts. Red dashed lines represent \|∆β\| > 0.2. (C,D). Comparison of hypoxia-induced gene expression signatures identiﬁed in gastric CAMs, ATMs, and NTMs. (C) Venn diagram showing the overlap between signiﬁcantly changed genes (limma p-value < 0.05) in response to hypoxia in CAM, ATM, and NTM cells. The common intersection represents a universal hypoxia-induced gene signature. Unique CAM-, ATM-, and NTM- hypoxia-induced gene signatures are highlighted in bold. (D) Genes universally changed in the same direction under hypoxia across all three populations of gastric myoﬁbroblasts (CAMs, ATMs, and NTMs, limma FDR p-value < 0.05). Figure 3. Hypoxia-induced gene expression signatures are not conferred by associated changes in DNA methylation. (A,B) Comparative global DNA methylation profiles of primary gastric myofibroblasts following exposure to hypoxic or normoxic conditions. (A) Volcano plot showing significance versus Δβ values for 424383 methylation probes in the three hypoxia (CAM, ATM, and NTM; n = 9) vs. normoxia (CAM, ATM and NTM; n = 9) comparisons. (B) Scatter plot representing mean β values for normoxic (CAM, ATM and NTM; n = 9) and hypoxic (CAM, ATM, and NTM; n = 9) myofibroblasts. Red dashed lines represent \|Δβ\| > 0.2. C,D. Comparison of hypoxia-induced gene expression signatures identified in gastric CAMs, ATMs, and NTMs. (C) Venn diagram showing the overlap between significantly changed genes (limma p-value < 0.05) in response to hypoxia in CAM, ATM, and NTM cells. The common intersection represents a universal hypoxia-induced gene signature Unique CAM- ATM- and NTM- hypoxia-induced gene signatures are highlighted in bold Figure 3. Hypoxia-induced gene expression signatures are not conferred by associated changes in D methylation. (A,B) Comparative global DNA methylation proﬁles of primary gastric myoﬁbrobla following exposure to hypoxic or normoxic conditions. 2.2.1. Differential Hypoxia-Induced Myoﬁbroblast Phenotypes Are Not Due to Changes in DNA Methylation Comparison of hypoxia-induced gene expression signatures identiﬁed in gastric CAMs, ATMs, and NTMs. (C) Venn diagram showing the overlap between signiﬁcantly changed genes (limma p-value < 0.05) in response to hypoxia in CAM, ATM, and NTM cells. The common intersection represents a universal hypoxia-induced gene signature. Unique CAM-, ATM-, and NTM- hypoxia-induced gene signatures are highlighted in bold. (D) Genes universally changed in the same direction under hypoxia across all three populations of gastric myoﬁbroblasts (CAMs, ATMs, and NTMs, limma FDR p-value < 0.05). Cancers 2019, 11, 263 7 of 18 7 of 18 2.2.2. Hypoxia-Induced Gene Expression Signatures 2.2.2. Hypoxia-Induced Gene Expression Signatures In general, cells appear to respond to hypoxia via a common HIF-1α mediated mechanism [18,19]; therefore, a subset of common hypoxia-induced gene expression changes may be expected in all populations of myoﬁbroblasts. However, since the net functional response of each population of myoﬁbroblasts (CAMs, ATMs, or NTMs) is different, it is likely that reported variations in epigenetic programming between different myoﬁbroblast populations [6] may confer differences in gene expression, and functional responses to hypoxic conditions. Differential gene expression analyses identiﬁed 702 genes that are universally changed under hypoxia in all myoﬁbroblast populations (Figure 3C), including a subset of genes that exhibit conserved directional changes (p-value < 0.05) across all myoﬁbroblast types (Figure 3D). Amongst the most upregulated genes are AK4, PFKFB3, BNIP3, and NDRG1, all of which show around 4-fold increases in expression under hypoxic conditions. In comparison, genes displaying the largest down-regulation in response to hypoxia are GANAB, SLC3A2, NQO1, and AIMP2 (FC < −2). To identify signalling pathways that are enriched in the group of 702 universal hypoxia-induced genes, gene ontology (GO) and gene set enrichment (GSEA) analyses were performed. Results from GO term analyses revealed glycolytic processes and metabolic processes to be the most signiﬁcantly represented (Figure S2). Reassuringly, GSEA analysis detected strong enrichment of gene sets characteristic for hypoxic cells, with ‘hallmark hypoxia’ and ‘hallmark glycolysis’ having the highest normalised enrichment scores. Notably, genes associated with those categories were almost exclusively grouped in the fraction of hypoxia up-regulated genes (Figure S3 and Table S1). 2.2.3. CAMs, ATMs, and NTMs Express Unique Gene Signatures Under Hypoxia Although common patterns of hypoxia-induced gene expression were observed in all populations of gastric myoﬁbroblasts, functional assays conﬁrmed that each population conferred differential effects on cancer cell migration and proliferation following exposure to hypoxic conditions. To investigate the molecular basis of these functional differences, subsets of unique hypoxia-induced genes were identiﬁed in each myoﬁbroblast population. In total, 894 genes were found to be uniquely changed in hypoxic CAMs, 1171 in hypoxic NTMs, and 1073 in hypoxic ATMs (Figure 3C). Each of these distinct conditional response signatures was then subjected to functional enrichment analysis using GO (Table S2), GSEA (Figure S4 and Table S3), and ingenuity pathway analysis (IPA, Figure S5). Overall, these analyses revealed unique transcriptional responses to hypoxia across different populations of myoﬁbroblasts. Most notably, upregulation of ‘cholesterol biosynthesis’ and ‘fatty acid metabolism’ emerged as a unique feature of hypoxic CAMs, while GSEA showed enrichment for ‘hallmark cholesterol homeostasis’ (Figure S4A). In addition, IPA revealed an upregulation of nearly all components of the ‘superpathway of cholesterol biosynthesis’ (Figure S5). These data strongly suggest that hypoxia enhances production of cholesterol in gastric CAMs, which may contribute to the energy metabolism of adjacent cancer cells. Notably, in patient-matched ATMs, low oxygen concentration uniquely induced the expression of genes involved in the G2/M cell cycle checkpoint, mitotic spindle assembly, and integrin signalling (Figures S4B and S5, Tables S2 and S3). Finally, analysis of the NTM unique gene expression signature showed that these genes simply contribute to signalling pathways related to hypoxia response and xenobiotic metabolism (Figures S4C and S5, Tables S2 and S3). The IPA downstream effects analysis, which facilitates the prediction of functions and processes that are expected to be increased or decreased based on changes in gene expression, also complements these observations (Figure S6). Having identiﬁed prominent population-speciﬁc differences in myoﬁbroblast responses to hypoxic conditions, it was important to investigate if factors secreted by hypoxic CAMs and NTMs could be predicted, based on changes in gene expression. Therefore, an IPA downstream effects analysis was performed for CAM-hypoxia vs. CAM-normoxia and NTM-hapoxia vs. NTM-normoxia comparisons. The results were then ﬁltered to include only genes that were likely to encode extracellular proteins that may be involved in cell migration and proliferation. 2.2.3. CAMs, ATMs, and NTMs Express Unique Gene Signatures Under Hypoxia Figure 4. Unique CAMs and NTMs hypoxia signatures may explain differential effects on cancer cell phenotype. (A,B). Predicted pro-migratory and pro-proliferative factors secreted by hypoxic CAMs and hypoxic NTMs. (A). Extracellular molecules expressed by hypoxic CAMs. (B). Extracellular molecules expressed by hypoxic NTMs. Molecules displayed in boxes are uniquely expressed in hypoxic CAM or hypoxic NTM. (C,D). Volcano plots of differentially secreted proteins in (C). CAM-hypoxic-CM vs. CAM-ctrl-CM and (D). NTM-hypoxic-CM vs. NTM-ctrl-CM comparisons. Proteins highlighted in red were annotated as secreted/extracellular or identiﬁed in exosomes based on MetazSecKB, Matrisome, and ExoCarta searches. Proteins above the line (p-value < 0.05) are considered to be differentially secreted between the given conditions. 2.2.3. CAMs, ATMs, and NTMs Express Unique Gene Signatures Under Hypoxia This procedure identiﬁed 66 pro-migratory 8 of 18 Cancers 2019, 11, 263 and pro-proliferative proteins that are likely to affect the observed cancer cell phenotype in response to CAM- and NTM-hypoxic-CM (Figure 4A,B). identified 66 pro-migratory and pro-proliferative proteins that are likely to affect the observed cancer cell phenotype in response to CAM- and NTM-hypoxic-CM (Figure 4A,B). Figure 4. Unique CAMs and NTMs hypoxia signatures may explain differential effects on cancer cell phenotype. A,B. Predicted pro-migratory and pro-proliferative factors secreted by hypoxic CAMs and hypoxic NTMs. A. Extracellular molecules expressed by hypoxic CAMs. B. Extracellular molecules expressed by hypoxic NTMs. Molecules displayed in boxes are uniquely expressed in hypoxic CAM or hypoxic NTM. C,D. Volcano plots of differentially secreted proteins in C. CAM- hypoxic-CM vs. CAM-ctrl-CM and D. NTM-hypoxic-CM vs. NTM-ctrl-CM comparisons. Proteins highlighted in red were annotated as secreted/extracellular or identified in exosomes based on MetazSecKB, Matrisome, and ExoCarta searches. Proteins above the line (p-value < 0.05) are considered to be differentially secreted between the given conditions. Figure 4. Unique CAMs and NTMs hypoxia signatures may explain differential effects on cancer cell phenotype. (A,B). Predicted pro-migratory and pro-proliferative factors secreted by hypoxic CAMs and hypoxic NTMs. (A). Extracellular molecules expressed by hypoxic CAMs. (B). Extracellular molecules expressed by hypoxic NTMs. Molecules displayed in boxes are uniquely expressed in hypoxic CAM or hypoxic NTM. (C,D). Volcano plots of differentially secreted proteins in (C). CAM-hypoxic-CM vs. CAM-ctrl-CM and (D). NTM-hypoxic-CM vs. NTM-ctrl-CM comparisons. Proteins highlighted in red were annotated as secreted/extracellular or identiﬁed in exosomes based on MetazSecKB, Matrisome, and ExoCarta searches. Proteins above the line (p-value < 0.05) are considered to be differentially secreted between the given conditions. 2 3 Analysis of Hypoxia Induced CAM and NTM Secretomes Figure 4. Unique CAMs and NTMs hypoxia signatures may explain differential effects on cancer cell phenotype. A,B. Predicted pro-migratory and pro-proliferative factors secreted by hypoxic CAMs and hypoxic NTMs. A. Extracellular molecules expressed by hypoxic CAMs. B. Extracellular molecules expressed by hypoxic NTMs. Molecules displayed in boxes are uniquely expressed in hypoxic CAM or hypoxic NTM. C,D. Volcano plots of differentially secreted proteins in C. CAM- hypoxic-CM vs. CAM-ctrl-CM and D. NTM-hypoxic-CM vs. NTM-ctrl-CM comparisons. Proteins highlighted in red were annotated as secreted/extracellular or identified in exosomes based on MetazSecKB, Matrisome, and ExoCarta searches. Proteins above the line (p-value < 0.05) are considered to be differentially secreted between the given conditions. y g 2.3. Analysis of Hypoxia-Induced CAM and NTM Secretomes 2.3. Analysis of Hypoxia-Induced CAM and NTM Secretomes In order to compare the profile of secreted proteins released by gastric myofibroblasts in response to hypoxia, conditioned media from CAMs and NTMs were profiled using liquid chromatography-mass spectrometry (LC-MS). In total, four conditions were examined: (i) CAM-ctrl- CM; (ii) CAM-hypoxic-CM; (iii) NTM-ctrl-CM; and (iv) NTM-hypoxic-CM. In each case, CM was collected after 72 h exposure to hypoxic (1% O2) or normoxic (21% O2) conditions. Altogether, 1169 protein groups were identified (FDR p-value < 0.01) and quantified across all samples (n = 12) using the MaxQuant computational platform. A core of 702 proteins was detected in all CM samples. These were subjected to gene ontology (GO) enrichment analysis using the PANTHER classification system [20] i d if h i ll l l i Th id ifi d i i ifi l i h d i In order to compare the proﬁle of secreted proteins released by gastric myoﬁbroblasts in response to hypoxia, conditioned media from CAMs and NTMs were proﬁled using liquid chromatography-mass spectrometry (LC-MS). In total, four conditions were examined: (i) CAM-ctrl- CM; (ii) CAM-hypoxic-CM; (iii) NTM-ctrl-CM; and (iv) NTM-hypoxic-CM. In each case, CM was collected after 72 h exposure to hypoxic (1% O2) or normoxic (21% O2) conditions. Altogether, 1169 protein groups were identiﬁed (FDR p-value < 0.01) and quantiﬁed across all samples (n = 12) using the MaxQuant computational platform. A core of 702 proteins was detected in all CM samples. These were subjected to gene ontology (GO) enrichment analysis using the PANTHER classiﬁcation system [20] in order to verify their cellular location. The identiﬁed proteins were signiﬁcantly 9 of 18 Cancers 2019, 11, 263 enriched in terms of annotation for extracellular region (p = 4.59 × 10−227) and extracellular vesicle (p = 1.74 × 10−200). The other top 10 most signiﬁcantly enriched GO terms were also associated with extracellular structures, conﬁrming that our experimental strategy worked. In addition, the Metazoa Secretome and Subcellular Proteome Knowledge Base [21], MatrisomeDB [22], and ExoCarta [23] were used to further annotate proteins identiﬁed in CAM or NTM secretomes. Overall, 76% were either known or predicted to be extracellular (Figure S7). 2.4. Gene Expression Proﬁles Enable Prediction of Secreted Factors Following an analysis of variance (ANOVA) test, a number of differentially secreted proteins were identiﬁed: 43 in the CAM-ctrl-CM vs. CAM-hypoxic-CM comparison (Figure 4C) and 225 in NTM-ctrl-CM vs. NTM-hypoxic-CM comparison (Figure 4D). However, only 14 (5%) of all signiﬁcantly changed proteins were altered in both hypoxic NTM and CAM secretomes (Figure S8). This reinforced our previous ﬁndings that different populations of myoﬁbroblasts display unique molecular signatures in response to environmental changes. Signiﬁcantly, proteomics analysis also conﬁrmed some conditional secretome signatures that were predicted from the analysis of corresponding gene expression proﬁles (Figure 4A,B). In particular, gelsolin (GSN), vascular endothelial growth factor A (VEGFA), and glucose-6-phosphate isomerase (GPI) were all veriﬁed at protein level to be differentially secreted by both hypoxic CAMs and NTMs (Figure 5A). In addition, hypoxic NTMs increased secretion of lysyl oxidase (LOX), insulin-like growth factor-binding protein 3 (IGFBP3), insulin-like growth factor-binding protein 6 (IGFBP6), and angiopoietin-related protein 4 (ANGPTL4). Furthermore, secretion of those proteins was not signiﬁcantly altered in hypoxic CAMs (Figure 5B,C), as predicted by IPA. This demonstrates that integration of multiple omics technologies enables validation of results, while also facilitating biologically relevant predictions. Another ﬁnding in concordance with previous gene expression studies relates to the potential biological effects of the secreted proteins. IPA downstream effects analysis indicated that cell death and necrosis may decrease in response to hypoxia-induced changes in CAM secretome composition. Furthermore, proliferation, invasion of tumour cell lines, and angiogenesis were predicted to increase in response to imposed changes in the NTM hypoxia-induced secretome (Figure S9). Notably, proteins secreted by hypoxic NTMs were predicted to be regulated by HIF-1α activation (z-score = 2.303, p = 4.76 × 10−9), as revealed by IPA upstream regulator analysis. These hypoxic NTM secreted proteins positively affect angiogenesis, migration of endothelial cells, and proliferation of tumour cell lines (Figure S10). These results provide new insights into the potential mechanisms by which hypoxia may enable stromal myoﬁbroblasts to promote cancer cell proliferation and migration as well as highlighting the molecular components involved in mediating these processes. 2.5. Secretion Is Not Strongly Regulated by Transcriptome Changes Although likely secreted factors can be predicted from gene expression data, the extent to which transcriptomic proﬁles reﬂect true patterns of protein expression or secretion remains uncertain. Nevertheless, on a simplistic level, it is possible that in some cases, increased mRNA expression may lead to higher concentrations of the encoded proteins and enhanced levels of secretion, or vice-versa. To identify subsets of proteins which show this correlation, fold changes of differentially expressed genes were compared with corresponding measurements of secreted proteins. For each comparison, a Pearson correlation coefﬁcient (r) was calculated and the signiﬁcance of the relationship was assessed by obtaining the corresponding p-value. Overall, a statistically signiﬁcant correlation was observed between these two variables. In CAMs, the transcriptome showed a weaker yet signiﬁcant correlation with the secretome (r = 0.42), while in NTMs, the co-regulation was substantially higher (r = 0.6) (Figure 5D). Furthermore, in both cases, the correlation was positive, indicating that on average, increases in transcript concentration lead to greater protein secretion in response to hypoxia. In addition, the magnitude of the changes was also similar. 10 of 18 Cancers 2019, 11, 263 Figure 5. Gene expression profiles enable prediction of secreted factors. A. Differential expression and secretion of gelsolin (GSN), vascular endothelial growth factor A (VEGFA), and glucose-6-phosphate isomerase (GPI) in hypoxic CAMs and hypoxic NTMs compared to respective control-normoxic myofibroblasts; p-value < 0.05. B. Differential expression and secretion of angiopoietin-related protein 4 (ANGPTL4), insulin-like growth factor-binding protein 3 (IGFBP3), insulin-like growth factor- binding protein 6 (IGFBP6), and thrombospondin-2 (THBS2) in gastric CAMs and NTMs under hypoxia and normoxia; p-value <0.05; checked–proteins detected in respective comparisons but not Figure 5. Gene expression proﬁles enable prediction of secreted factors. (A). Differential expression and secretion of gelsolin (GSN), vascular endothelial growth factor A (VEGFA), and glucose-6-phosphate isomerase (GPI) in hypoxic CAMs and hypoxic NTMs compared to respective control-normoxic myoﬁbroblasts; p-value < 0.05. (B). Differential expression and secretion of angiopoietin-related protein 4 (ANGPTL4), insulin-like growth factor-binding protein 3 (IGFBP3), insulin-like growth factor-binding protein 6 (IGFBP6), and thrombospondin-2 (THBS2) in gastric CAMs and NTMs under hypoxia and normoxia; p-value < 0.05; checked–proteins detected in respective comparisons but not identiﬁed as differentially secreted (p-value > 0.05). (C). Differential expression and secretion of enzyme lysyl oxidase (LOX) in gastric CAMs and NTMs under hypoxia and normoxia. 2.5. Secretion Is Not Strongly Regulated by Transcriptome Changes LOX mRNA transcript was not identiﬁed as differentially expressed in hypoxic CAMs compared to control-normoxic CAMs (p-value > 0.05), whereas LOX protein (checked) was detected in CAM-hypoxic-CM and CAM-ctrl-CM; however, it was not identiﬁed as signiﬁcant in differential secretome analysis (p-value > 0.05). (D). Integration of secretome and gene expression data for CAM hypoxia vs. CAM normoxia and NTM hypoxia vs. NTM normoxia. The dashed line represents the best linear ﬁt between the two variables. Grey points indicate that from the mRNA-protein pair, only the transcript was determined to be signiﬁcantly changing (p-value < 0 05) and red points that both protein and transcript were signiﬁcantly altered Figure 5. Gene expression profiles enable prediction of secreted factors. A. Differential expression and secretion of gelsolin (GSN), vascular endothelial growth factor A (VEGFA), and glucose-6-phosphate isomerase (GPI) in hypoxic CAMs and hypoxic NTMs compared to respective control-normoxic myofibroblasts; p-value < 0.05. B. Differential expression and secretion of angiopoietin-related protein 4 (ANGPTL4), insulin-like growth factor-binding protein 3 (IGFBP3), insulin-like growth factor- binding protein 6 (IGFBP6), and thrombospondin-2 (THBS2) in gastric CAMs and NTMs under hypoxia and normoxia; p-value <0.05; checked–proteins detected in respective comparisons but not Figure 5. Gene expression proﬁles enable prediction of secreted factors. (A). Differential expression and secretion of gelsolin (GSN), vascular endothelial growth factor A (VEGFA), and glucose-6-phosphate isomerase (GPI) in hypoxic CAMs and hypoxic NTMs compared to respective control-normoxic myoﬁbroblasts; p-value < 0.05. (B). Differential expression and secretion of angiopoietin-related protein 4 (ANGPTL4), insulin-like growth factor-binding protein 3 (IGFBP3), insulin-like growth factor-binding protein 6 (IGFBP6), and thrombospondin-2 (THBS2) in gastric CAMs and NTMs under hypoxia and normoxia; p-value < 0.05; checked–proteins detected in respective comparisons but not identiﬁed as differentially secreted (p-value > 0.05). (C). Differential expression and secretion of enzyme lysyl oxidase (LOX) in gastric CAMs and NTMs under hypoxia and normoxia. LOX mRNA transcript was not identiﬁed as differentially expressed in hypoxic CAMs compared to control-normoxic CAMs (p-value > 0.05), whereas LOX protein (checked) was detected in CAM-hypoxic-CM and CAM-ctrl-CM; however, it was not identiﬁed as signiﬁcant in differential secretome analysis (p-value > 0.05). (D). Integration of secretome and gene expression data for CAM hypoxia vs. CAM normoxia and NTM hypoxia vs. NTM normoxia. The dashed line represents the best linear ﬁt between the two variables. 3. Discussion Hypoxia is an important microenvironmental factor in the development and metastasis of many solid tumours. During tumour development, both cancer and stromal cells are exposed to hypoxic conditions. This study provides novel evidence that hypoxia induces differences in the functional effects that gastric CAMs and NTMs exert on cancer cell migration and proliferation. In particular, hypoxia signiﬁcantly increases the ability of CAMs to promote the migration of cancer cells, while also inducing pro-tumorigenic CAM-like properties in gastric NTMs. Genome-wide DNA methylation proﬁling of hypoxia-treated CAMs and NTMs showed that although CAMs and NTMs have distinctive patterns of DNA methylation [6], these characteristic proﬁles were not signiﬁcantly altered by hypoxic conditioning, suggesting that unique transcriptional responses to hypoxia observed among different populations of gastric myoﬁbroblasts (CAMs, ATMs, NTMs) most likely stem from differential retained epigenetic backgrounds. As such, hypoxia, as a single microenvironmental factor, is unlikely to impose CAM-like DNA methylation patterns in normal tissue myoﬁbroblast (NTMs). y p y Interestingly, upregulation of cholesterol biosynthesis and fatty acid metabolism emerged as a unique feature of hypoxic CAMs, with all components of the “superpathway” for cholesterol biosynthesis showing signiﬁcant upregulation, indicating that hypoxia may enhance cholesterol production in gastric CAMs. In this context, malignant cells have previously been shown to increase lipolysis in adipocytes, which then secrete fatty acids that are then taken up by neighbouring cancer cells to enhance energy production [24]. Data from this study suggest that under hypoxic conditions, gastric CAMs may serve a similar function. In particular, coupling of energy metabolism between CAMs and cancer cells may be mediated by fatty acid binding proteins, such as FABP1 and FABP3, which were also upregulated in hypoxic CAMs. Finally, upregulation of cholesterol biosynthesis under hypoxia in CAMs may serve as an adaptive mechanism that protects CAMs from oxidative damage. Activation of de novo lipogenesis in cancer cells leads to increased membrane lipid saturation, resulting in higher levels of saturated and monounsaturated phospholipids, which may protect cancer cells from free radicals and chemotherapeutics [25]. Transcriptional analysis of the unique gene set identiﬁed in hypoxic NTMs shows a more classic response, mainly composed of genes known to contribute to hypoxic signalling pathways, hypoxia response, and xenobiotic metabolism. The IPA downstream effect analysis performed in this study predicts distinct biological effects of hypoxia among different gastric myoﬁbroblast populations. 2.5. Secretion Is Not Strongly Regulated by Transcriptome Changes Grey points indicate that from the mRNA-protein pair, only the transcript was determined to be signiﬁcantly changing (p-value < 0.05), and red points that both protein and transcript were signiﬁcantly altered (p-value < 0.05). r–Pearson correlation, p–p-value indicating the signiﬁcance of the correlation. Cancers 2019, 11, 263 11 of 18 11 of 18 3. Discussion Signiﬁcantly, activation of autophagy emerged as a key CAM-speciﬁc process that was enhanced in response to hypoxia, whereas processes such as organisation of organelle, growth of microtubules, and aneuploidy of cells appeared to be activated only in hypoxic ATMs, while expression and transcription of RNA were the most signiﬁcantly activated processes in hypoxic NTMs. Interestingly, processes such as mitosis, angiogenesis, invasion of cells, growth of axons, neoplasia of tumour cell lines, and glycolysis of cells were all predicted to be most active in hypoxic NTMs. Secretome analysis conﬁrmed some of the predictions based on hypoxia-induced CAM and NTM gene expression proﬁles. Interestingly, lysyl oxidase (LOX), among others, was veriﬁed at the protein level as an NTM unique hypoxia-induced secreted protein. LOX is an extracellular amine oxidase that post-translationally modiﬁes collagens and elastin in the extracellular matrix, catalysing the covalent crosslinking of ﬁbres [26], thus contributing to hypoxia-induced metastasis. LOX is highly expressed in hypoxic tumour cells, including breast cancer, where it promotes extracellular matrix remodelling in the lungs, leading to the formation of a metastatic niche [27]. Comparative analysis of secretomes and gene expression proﬁles revealed that both expression and secretion of LOX is increased in CAMs compared to NTMs under normal conditions. However, when oxygen is limited, LOX is also induced in NTMs, both at the mRNA and protein level. Comparative proﬁling of the secretomes of hypoxic CAMs, compared to control CAMs or hypoxic and control NTMs, shows that hypoxia has a signiﬁcantly greater impact on protein secretion in NTMs than CAMs. This observation is also consistent with IPA downstream effect analysis, which predicts Cancers 2019, 11, 263 12 of 18 that expression and transcription of RNA is the most signiﬁcantly activated processes in hypoxic NTMs. Taken together, these data suggest that CAMs are somehow less responsive to low oxygen levels then NTMs. Proteins differentially secreted by NTMs in response to hypoxia were predicted to be regulated by HIF-1α activation and are known to enhance tumour cell proliferation, invasion, cell movement, angiogenesis, vasculogenesis, and the migration of endothelial cells. Signiﬁcantly, these data are consistent with experimental data presented in this study, showing increased cancer cell migration and proliferation in response to NTM-hypoxic-CM compared to NTM-ctrl-CM. By contrast, proteins differentially secreted by hypoxic CAMs, compared to control CAMs, were predicted to reduce cell death via necrosis or apoptosis. 4.2. Gastric Cancer Cell Culture Human gastric adenocarcinoma cells (AGS) were cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 1% penicillin–streptomycin, and 1% antibiotic–antimitotic solution. The AGS cells were incubated at 37 ◦C in a humidiﬁed atmosphere with 5% CO2 and were passaged or growth media were changed approximately every 48–72 h. In all experiments, cells were used between passages 18 and 28. 3. Discussion Taken together, these results concur with gene expression analysis of hypoxia-induced NTM- and CAM-speciﬁc gene signatures. yp p g g In summary, this study is the ﬁrst to perform comparative multi-omics proﬁling to investigate the global effects of hypoxia on DNA methylation, gene expression, and protein secretion in populations of primary gastric myoﬁbroblasts, derived from either cancer (CAMs), pre-neoplastic (ATMs), or normal (NTMs) tissue. Data presented in this study provide new insight into the differential molecular responses of myoﬁbroblasts to hypoxic conditions within the gastric tumour microenvironment. The integrated multi-omic data sets from this work will act as a resource to inform future studies into the molecular mechanisms of tumour progression, while also facilitating comparative analysis of the effects of hypoxia on myoﬁbroblast function in the microenvironment of other solid tumours. 4.1. Primary Gastric Myoﬁbroblasts Human primary gastric myoﬁbroblasts were derived as previously described [28]. In each case, isolated myoﬁbroblast populations were analysed to conﬁrm stellate/spindle-shaped morphology and consistent expression of both α-SMA and vimentin, as shown previously [5]. Primary myoﬁbroblast cultures were maintained in Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 1% penicillin–streptomycin, 1% antibiotic–antimycotic, and 1% non-essential amino acid solution. Medium was replaced every 48–60 h. In all cases, myoﬁbroblasts were not maintained beyond passage 12. 4.3. Hypoxic Conditioned Media Preparation In order to generate CAM or NTM hypoxic conditioned media (CAM-hypoxic-CM or NTM-hypoxic-CM) and CAM or NTM normoxic conditioned media (CAM-ctrl-CM or NTM-ctrl-CM), 5 × 105 of selected myoﬁbroblast cell lines were seeded in 75 cm2 ﬂasks and left attached for 24 h in growth media. The next day, the media were replaced, and cells were put either to normoxia (21% O2) or hypoxia (1% O2) for 48 h incubation in serum-supplemented DMEM. After 48 h, the cells were washed three times in 1× PBS to get rid of any serum-derived factors, and 13 mL freshly prepared serum-free DMEM supplemented with 1% penicillin–streptomycin, 1% antibiotic–antimycotic, and 1% non-essential amino acid solution was added to the cells. The serum-free media were incubated with the cells for 24 h in normal (21% O2) and hypoxic (1% O2) conditions. The following day, myoﬁbroblast ctrl-CM and hypoxic-CM were collected and centrifuged at 800g for 7 min to remove cell debris. Cancers 2019, 11, 263 13 of 18 The freshly prepared CAM or NTM hypoxic-CM and ctrl-CM were immediately used for cancer cell migration and proliferation assays. The freshly prepared CAM or NTM hypoxic-CM and ctrl-CM were immediately used for cancer cell migration and proliferation assays. 4.6. Integrated Multi-Omics Myoﬁbroblast Proﬁling Gastric cancer patient-matched CAMs and ATMs (n = 3) as well as unrelated NTMs (n = 3) were cultured in parallel for integrated genome-wide DNA methylation, gene expression, and secretome proﬁling. In order to generate myoﬁbroblast conditioned media, 500 × 103 of selected myoﬁbroblast cell lines were seeded in 75 cm2 ﬂasks. Growth media were replaced after 24 h and cells were incubated under normoxic (21% O2) or hypoxic (1% O2) conditions for 48 h. After this initial incubation, cells were washed three times in 1× PBS to remove serum-derived proteins, and 13 mL of freshly prepared serum-free DMEM supplemented with 1% penicillin–streptomycin, 1% antibiotic–antimycotic, and 1% non-essential amino acid solution was added and conditioned for 24 h under normoxic or hypoxic conditions. Conditioned media and cell lysates for DNA and RNA extraction were collected for use in Illumina Inﬁnium HumanMethylation450k and HumanHT-12v4 Expression arrays as well as LC-MS/MS secretome analysis. 4.4. Cancer Cell Migration Assay The effects of hypoxic-CAM and hypoxic-NTM conditioned media (CM) on gastric cancer cells migration were measured in vitro using trans-well Boyden chamber assay (SLS; cat. no. 354578). Brieﬂy, 1 × 104 AGS cells in 500 µL of serum-free DMEM medium were added to the BioCoat Control Inserts with 8µm pore PET membrane (upper chambers). The lower chambers contained either 750 µL serum-free media, or myoﬁbroblast CM to serve as a chemoattractant. Cells were incubated at 37 ◦C and allowed to migrate overnight. Thereafter, AGS cells were removed from the upper surface of the membrane by scrubbing with cotton swabs. Cells migrating through the membrane were ﬁxed and detected on the lower surface using Reastain Quick-Diff Kit (Reagena; cat. no. 102164, Toivala, Finland) and then examined under a bright-ﬁeld microscope. Values for cancer cell migration were obtained by counting ﬁfteen ﬁelds per membrane (20× objective) and represent an average of at least three independent membranes. 4.5. Cancer Cell Proliferation Assay The effects of hypoxic-CAM and hypoxic-NTM conditioned media (CM) on gastric cancer cell proliferation were assessed by incorporation of 5-ethynyl-2′-deoxyuridine (EdU) [29] and detected using the Click-iT EdU Alexa Fluor 488 Imaging Kit (Life Technologies; cat. no. C10337, California, CA, USA). Brieﬂy, 1 × 104 AGS cells were plated onto cover glasses in 24-well plate and left for 24 h at 37 ◦C to attach. The next day, growth media were replaced with serum-free DMEM supplemented with 1% penicillin–streptomycin and 1% antibiotic–antimycotic to synchronise the cells. Thereafter, the media were replaced with 1 ml of either serum-free media or myoﬁbroblast CM containing 10 µM EdU, and cells were incubated overnight at 37 ◦C, 5% CO2. Following incubation, the manufacturer’s protocol was applied to ﬁx and permeabilise the AGS cells and detect EdU incorporation. 4.7. Gene Ontology Enrichment Analysis Gene Ontology (GO) enrichment analysis [40] was performed on hypoxia-induced gene expression proﬁles identiﬁed in CAMs, ATMs, and NTMs using GOrilla (gene ontology enrichment analysis and visualization tool) [41]. The target genes were compared to the background gene set of all expressed and normalised genes from the HT-12v4 Illumina experiment. The enriched GO terms in individual CAM, ATM, and NTM hypoxia-induced gene expression proﬁles were discovered using a hypergeometric model, p-value < 0.001 was applied, and the results were corrected for multiple testing using the Benjamini and Hochberg method [34]. To reduce redundancy within the identiﬁed enriched lists of GO terms, web server REVIGO [42] was used. 4.6.3. Secretome Proﬁling Conditioned media from normoxic and hypoxic myoﬁbroblasts were centrifuged at 800 g for 7 min to remove cell debris and supernatants were aliquoted and stored at −80 ◦C prior to LC-MS/MS secretome analysis, which was performed by the Centre for Proteome Research (University of Liverpool, Liverpool, UK). Raw mass spectrometry data were analysed using MaxQuant software [36] with Andromeda [37] as a search engine against the reference human proteome (Uniprot proteome ID UP000005640, containing 42112 reviewed canonical and isoform protein entries, accessed 30 April 2015) and 123 potential contaminants. Protein quantiﬁcation was obtained using the MaxQuant intensity-based label free quantiﬁcation (LFQ) algorithm. Potential contaminants identiﬁed in the mass spectrometry data were manually examined, and proteins associated with foetal bovine serum (FBS) or trypsin were rejected. The LFQ algorithm extracts protein intensities form the raw data and performs appropriate normalisation steps to allow between sample comparisons and remove any systematic errors during data acquisition. For simplicity, the protein group was reported in all cases where a protein was identiﬁed by peptides that could not be assigned to individual protein unambiguously, for example, in cases of highly homologous proteins, or protein isoforms. On average, each protein was quantiﬁed with 7 unique peptides and 22% sequence coverage was achieved. To compare CAM and NTM secretomes from different microenvironmental conditions, four pair-wise comparisons were done using a one-way analysis of variance (ANOVA) test as implemented in DanteR software [38]. Proteins with a p-value < 0.05 were considered to be differentially secreted between the two conditions. The raw mass spectrometry secretome data and processing results from MaxQuant have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [39] with the identiﬁer PXD008104. 4.6.2. Illumina Inﬁnium HumanMethylation450k Array Genomic DNA for DNA methylation analysis using the Illumina Inﬁnium HumanMethylation450 BeadChip arrays was puriﬁed using a standard phenol/chloroform extraction method. DNA sample purity and extent of degradation were determined by spectrophotometry at 260/280 nm and gel electrophoresis on 1% agarose gel, respectively. DNA quantity was assessed using PicoGreen ﬂuorimetry (Life Technologies, cat. no. Q-33130, California, CA, USA). Raw DNA methylation data were processed and analysed using Bioconductor package RnBeads version 0.99.17 [35] as described previously [6]. 4.6.1. Illumina Human HT-12 v4 Array Total RNA for gene expression analysis using the Illumina HumanHT-12v4 Expression BeadChip arrays was puriﬁed using miRNeasy Mini Kit (Qiagen, cat. no. 217004, Hilden, Germany) according to manufacturer’s instructions. RNA sample quality and quantity were assessed by spectrophotometry at 260/280 nm. The Bioconductor package lumi (version 2.18.0) [30] was used to import the raw intensities data into R and perform background correction, variance stabilisation transformation [31], robust spline normalisation, and subsequent quality control. A biological replicate of one of the samples was included to assess the reproducibility of sample preparation (R2 = 0.9933573). Probes with a detection p-value < 0.01 across all samples were considered non-detectable and were removed from analysis. Un-annotated probes were also removed, restricting the subsequent analysis to 18090 14 of 18 Cancers 2019, 11, 263 14 of 18 probes (corresponding to 13381 genes; GEO accession number: GSE125177). To identify differentially expressed transcripts in (i) CAM hypoxia vs. CAM normoxia, (ii) NTM hypoxia vs. NTM normoxia, and (iii) ATM hypoxia vs. ATM normoxia comparisons, the Bioconductor package limma [32,33] was used. The Benjamini and Hochberg method [34] was used to control for false discovery rate. 4.9. Ingenuity Pathway Analysis Ingenuity pathway analysis (IPA) software was used to analyse and interpret the hypoxia-induced gene expression proﬁles identiﬁed in CAMs, ATMs, and NTMs and hypoxia-induced CAM and NTM secretome proﬁles. Gene expression proﬁles were compared against an IPA predeﬁned Illumina HT-12v4 reference set, whereas secretome proﬁles were compared against an Ingenuity Pathway Analysis knowledge base (IKB). For gene expression data, an IPA canonical pathway and downstream effects analyses were performed. For secretome data, IPA downstream effects and upstream regulator analyses were performed. In each case, p-value was assigned using Fisher’s exact test, which indicates the probability of overlap between the pathway/phenotype and input genes. 4.10. R/Bioconductor R statistical software (version 3.1.2) and Bioconductor [45,46] were used to process and analyse gene expression, DNA methylation, and secretome data. 4.8. Gene Set Enrichment Analysis Gene set enrichment analysis (GSEA v5.0) [43,44] was used to characterise and interpret the identiﬁed unique-CAM, ATM, or NTM and universal hypoxia-induced gene expression proﬁles. 15 of 18 Cancers 2019, 11, 263 15 of 18 The Molecular Signatures Database (MSigDB v5.0) [44] that contains collection of biologically predeﬁned gene sets was used to determine statistically enriched gene sets present in the analysed gene expression data. The identiﬁed unique-CAM, ATM, or NTM and universal hypoxia-induced gene expression proﬁles were separated into two phenotypes for GSEA: Hypoxia and normoxia. For gene list ranking, multiple probes matching the same gene were sorted according to p-value, and the probe with the lowest p-value was retained for the analysis. Genes were ranked using the provided signal-to-noise ranking statistic, and GSEA was run using default weighted enrichment statistics and evaluated for statistical signiﬁcance by comparison to results obtained using 1000 random permutations of each gene set. Default settings were used for all other GSEA parameters. Author Contributions: Conceptualisation, H.N. and C.M.S.; formal analysis, H.N. and A.F.J.; investigation, H.N.; resources, A.V.; data curation, H.N.; writing—original draft preparation, H.N.; writing—review and editing, H.N., A.F.J., C.M.S. and A.V.; visualisation, H.N.; supervision, C.M.S.; funding acquisition, C.M.S. and A.V. References 1. 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[CrossRef] [PubMed] 14. Gilkes, D.M.; Semenza, G.L.; Wirtz, D. Hypoxia and the extracellular matrix: Drivers of tumour metastasis. Nat. Rev. Cancer 2014, 14, 430–439. [CrossRef] [PubMed] 14. Gilkes, D.M.; Semenza, G.L.; Wirtz, D. Hypoxia and the extracellular matrix: Drivers of tumour metastasis. Nat. Rev. Cancer 2014, 14, 430–439. [CrossRef] [PubMed] 15. Dayan, F.; Mazure, N.M.; Brahimi-Horn, M.C.; Pouysségur, J. A dialogue between the hypoxia-inducible factor and the tumor microenvironment. Cancer Microenviron. 2008, 1, 53–68. [CrossRef] [PubMed] 16. Giaccia, A.J.; Schipani, E. Role of carcinoma-associated ﬁbroblasts and hypoxia in tumor progression. 5. Conclusions Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. Conclusions This study shows that patient-derived gastric cancer-associated myoﬁbroblasts (CAMs) exhibit unique changes in gene expression and protein secretion in response to hypoxic conditions. Although hypoxia increased production of factors involved in matrix remodelling in both CAMs and NTMs, upregulation of factors required for cholesterol production and metabolism was only seen in CAMs. These ﬁndings suggest that gastric CAMs, unlike patient-matched ATMs or NTMs, contribute to cancer cell growth and metabolism. Functional studies show hypoxia increases the ability of gastric CAMs to enhance cancer cell proliferation and migration, while also conferring pro-tumorigenic properties in non-cancer derived NTMs. Conditional multi-omics data from this study will provide a resource to facilitate future comparative studies to identify common mechanisms of tumour myoﬁbroblast interactions in other solid tumours. Supplementary Materials: Available online at http://www.mdpi.com/2072-6694/11/2/263/s1. Figure S1: Differential effects of CAM and NTM hypoxic conditioned media (CM) on AGS gastric cancer cell migration and proliferation, Figure S2: Universal changes induced by hypoxia in gastric CAMs, ATMs and NTMs, Figure S3: GSEA enrichment plots for the most signiﬁcantly enriched A. hallmark gene set and B. canonical pathway gene set, Table S1: GSEA result summary, Table S2: Gene ontology (GO) biological process (BP) enrichment for unique hypoxia-induced gene expression signatures identiﬁed in CAMs, ATMs or NTMs, Figure S4: GSEA enrichment plots for the most signiﬁcantly enriched hallmark gene sets, Table S3: GSEA result summary, Figure S5: IPA canonical pathways signiﬁcantly enriched in unique CAM, ATM and NTM hypoxia-induced gene expression proﬁles, Figure S6: Predicted biological effects of hypoxia on gastric CAMs, ATMs and NTMs, Figure S7: Venn diagram representation of database searches used to classify proteins identiﬁed in CAM and NTM conditioned media obtained from normoxia and hypoxia, Figure S8: Comparison of differentially secreted proteins identiﬁed in CAM-hypoxic-CM and NTM-hypoxic-CM compared to their respective normoxic-control-CM, Figure S9: Predicted biological effects of the hypoxia-induced CAM and NTM secretomes, Figure S10: Predicted activation of HIF-1α regulates expression of proteins secreted by hypoxic NTMs. Author Contributions: Conceptualisation, H.N. and C.M.S.; formal analysis, H.N. and A.F.J.; investigation, H.N.; resources, A.V.; data curation, H.N.; writing—original draft preparation, H.N.; writing—review and editing, H.N., A.F.J., C.M.S. and A.V.; visualisation, H.N.; supervision, C.M.S.; funding acquisition, C.M.S. and A.V. 16 of 18 16 of 18 Cancers 2019, 11, 263 Funding: This research was funded by the Cancer Research UK non-clinical training award [C35628/A12779]. 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https://openalex.org/W4385506340	https://pure.coventry.ac.uk/ws/files/74780547/Published.pdf	English	null	How to enable large format 4680 cylindrical lithium-ion batteries	Applied energy	2,023	cc-by	13,865	How to enable large format 4680 cylindrical lithium-ion batteries Li, S., Marzook, M. W., Zhang, C., Offer, G. J. & Marinescu, M. Published PDF deposited in Coventry University’s Repository Original citation: Li, S, Marzook, MW, Zhang, C, Offer, GJ & Marinescu, M 2023, 'How to enable large format 4680 cylindrical lithium-ion batteries', Applied Energy, vol. 349, 121548. https://dx.doi.org/10.1016/j.apenergy.2023.121548 DOI 10.1016/j.apenergy.2023.121548 ISSN 0306-2619 ESSN 1872-9118 Publisher: Elsevier This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) How to enable large format 4680 cylindrical lithium-ion batteries Li, S., Marzook, M. W., Zhang, C., Offer, G. J. & Marinescu, M. Published PDF deposited in Coventry University’s Repository Original citation: Li, S, Marzook, MW, Zhang, C, Offer, GJ & Marinescu, M 2023, 'How to enable large format 4680 cylindrical lithium-ion batteries', Applied Energy, vol. 349, 121548. https://dx.doi.org/10.1016/j.apenergy.2023.121548 DOI 10.1016/j.apenergy.2023.121548 ISSN 0306-2619 ESSN 1872-9118 Publisher: Elsevier This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) H I G H L I G H T S • Distributed 3D electro-thermal model built for cylindrical cells with true structure. • The ability to thermally manage cylindrical cells limits their maximum size. • Surface to volume ratio and inner structure are critical to cell performance. • Large cylindrical cells must be base cooled and have continuous tabs. • For small cylindrical cells side cooling is most efficient. A R T I C L E I N F O Keywords: Large format lithium-ion battery 4680 tabless cell Electro-thermal model Cell design Thermal management The demand for large format lithium-ion batteries is increasing, because they can be integrated and controlled easier at a system level. However, increasing the size leads to increased heat generation risking overheating. 1865 and 2170 cylindrical cells can be both base cooled or side cooled with reasonable efficiency. Large format 4680 cylindrical cells have become popular after Tesla filed a patent. If these cells are to become widely used, then understanding how to thermally manage them is essential. In this work, we create a model of a 4680 cy lindrical cell, and use it to study different thermal management options. Our work elucidates the comprehensive mechanisms how the hot topic ‘tabless design’ improves the performance of 4680 cell and makes any larger format cell possible while current commercial cylindrical cells cannot be simply scaled up to satisfy power and thermal performance. As a consequence, the model identifies the reason for the tabless cell's release: the thermal performance of the 4680 tabless cell can be no worse than that of the 2170 cell, while the 4680 tabless tab cell boasts 5.4 times the energy and 6.9 times the power. Finally, via the model, a procedure is proposed for choosing the thermal management for large format cylindrical cell for maximum performance. As an example, we demonstrate that the best cooling approach for the 4680 tabless cell is base cooling, while for the 2170 LG M50T cell it is side cooling. We conclude that any viable large format cylindrical cell must include a continuous tab (or ‘tabless’) design and be cooled through its base when in a pack. The results are of immediate interest to both cell manufacturers and battery pack designers, while the developed modelling and parameterization framework is of wider use for all energy storage system design. performance efficiency of LIBs throughout their lifetime. pi y g In recent years, large format lithium-ion batteries have been devel oped for applications such as electric vehicles [2–6]. Large format cells have a number of advantages over smaller form factors. Firstly, the number of interconnectors and control circuits in a battery pack can be reduced, avoiding unnecessary energy loss via voltage drop and dissi pative heat generation, as well as enhancing the system reliability [7]. How to enable large format 4680 cylindrical lithium-ion batteries Li, S., Marzook, M. W., Zhang, C., Offer, G. J. & Marinescu, M. Published PDF deposited in Coventry University’s Repository Original citation: Li, S, Marzook, MW, Zhang, C, Offer, GJ & Marinescu, M 2023, 'How to enable large format 4680 cylindrical lithium-ion batteries', Applied Energy, vol. 349, 121548. https://dx.doi.org/10.1016/j.apenergy.2023.121548 DOI 10.1016/j.apenergy.2023.121548 ISSN 0306-2619 ESSN 1872-9118 Publisher: Elsevier This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Li, S., Marzook, M. W., Zhang, C., Offer, G. J. & Marinescu, M. Applied Energy 349 (2023) 121548 How to enable large format 4680 cylindrical lithium-ion batteries Shen Li a,c,1, Mohamed Waseem Marzook a, Cheng Zhang b, Gregory J. Offer a,c, Monica Marinescu a,c,* a Department of Mechanical Engineering, Imperial College London, London SW7 2AZ, United Kingdom b Institute for Future Transport and Cities, Coventry University, Coventry CV1 5FB, United Kingdom c The Faraday Institution, Harwell Science and Innovation Campus, Didcot OX11 0RA, United Kingdom a Department of Mechanical Engineering, Imperial Available online 2 August 2023 0306-2619/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). * Corresponding author at: Department of Mechanical Engineering, Imperial College London, London SW7 2AZ, United Kingdom. E-mail address: monica.marinescu@imperial.ac.uk (M. Marinescu). 1 Present address: Rimac Technology R&D UK Limited, Coventry CV34 6RG, United Kingdom. https://doi.org/10.1016/j.apenergy.2023.121548 Received 31 January 2023; Received in revised form 14 June 2023; Accepted 1 July 2023 * Corresponding author at: Department of Mechanical Engineering, Imperial College London, London SW7 2AZ, United Kingdom. E-mail address: monica.marinescu@imperial.ac.uk (M. Marinescu). 1 Present address: Rimac Technology R&D UK Limited, Coventry CV34 6RG, United Kingdom. Available online 2 August 2023 0306-2619/© 2023 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.apenergy.2023.121548 Received 31 January 2023; Received in revised form 14 June 2023; Accepted 1 July 2023 1. Introduction and motivation Prior modelling [14] and experimental work [15] have shown that using more tabs for cylindrical cells acts to reduce heat generation and thermal gradients inside the cell. The continuous tab design has been predicted to efficiently reduce heat generation [14], however, due to manufacturing challenges, no commercial cell with continuous tab had been realized until recently. A patent [17] by Tesla Inc. introduces a solution for the manufacturing of large format tabless 4680 cylindrical cells with a novel ‘tabless’ design, essentially a continuous tab. Based on model predictions [14], this continuous tab design is expected to over come the thermal issues associated with large cylindrical cell size through reduced heat generation and increased heat dissipation. The benefit of tabless design on current distribution and temperature for 4680 cells has been demonstrated using a 2D electrochemical jellyroll model [18]. However, factors such as the effect of the current paths through the current collector, the connection between the metal can and the jellyroll and the chosen cooling strategy were not included. These considerations have been shown to be critical in assessing the viability and performance of cylindrical cells [13], but have not been assessed for the 4680 tabless cell. Secondly, the pack level energy and power density can be higher due to the reduced proportion of casing material [8]. However, a potential disadvantage of large format cells is overheating, when the importance of effective thermal management is overlooked. Without effective heat dissipation, higher internal temperatures and larger thermal gradients can develop inside larger cells [9,10], which can limit performance and accelerate degradation significantly [11]. Secondly, the pack level energy and power density can be higher due to the reduced proportion of casing material [8]. However, a potential disadvantage of large format cells is overheating, when the importance of effective thermal management is overlooked. Without effective heat dissipation, higher internal temperatures and larger thermal gradients can develop inside larger cells [9,10], which can limit performance and accelerate degradation significantly [11]. i The heat dissipation rate of a cell is strongly affected by its surface- to-volume ratio and thermal conductivity [12]. For cylindrical cells, the surface-to-volume ratio is inherently poor due to geometry, as it is inversely proportional to the radius. The thermal conductivity is strongly anisotropic, with a low thermal conductivity in the radial di rection [13]. 1. Introduction and motivation Lithium-ion batteries (LIBs) are a popular energy storage solution due to their high energy and power density, low self-discharge rate and long cycle life [1]. To further reduce both the economic and environ mental costs associated with LIBs, there is a strong need to improve the Applied Energy 349 (2023) 121548 S. Li et al. 1. Introduction and motivation Nomenclature Abbreviations Description CCC Cell cooling coefficient ECN Equivalent circuit network ESS Energy storage system EV Electric vehicle LIB Lithium-ion battery MSMD Multi-scale multi-dimensional NTGK Semi-empirical model named by Newman, Tiedemann, Gu and Kwon P2D Pseudo two-dimensional SoC State of charge Symbols Description (Unit) Abase Cell base area (m2) CCCbase Cell cooling coefficient under base cooling condition (W⋅K−1) C̃CCGN base Base cooling CCC normalized for geometry (W⋅K−1⋅m−3) C̃CCHG base Base cooling CCC normalized for heat generation (W⋅K−1⋅Ah−2⋅Ω-1) CCCside Cell cooling coefficient under side cooling condition (W⋅K−1) C̃CCGN side Side cooling CCC normalized for geometry (W⋅K−1⋅m−3) C̃CCHG side Side cooling CCC normalized for heat generation (W⋅K−1⋅Ah−2⋅Ω-1) CR C rate (h−1) hinsu Effective heat transfer coefficient for heat loss through L λlink q Q Qbase Qext Qgen Qside R R0 S1 S2 t Tavg Tmin Tmax Ttarget ΔT ΔTavg ΔTaxia ΔTgrad ΔTradi Nomenclature Abbreviations Description CCC Cell cooling coefficient ECN Equivalent circuit network ESS Energy storage system EV Electric vehicle LIB Lithium-ion battery MSMD Multi-scale multi-dimensional NTGK Semi-empirical model named by Newman, Tiedemann, Gu and Kwon P2D Pseudo two-dimensional SoC State of charge Symbols Description (Unit) Abase Cell base area (m2) CCCbase Cell cooling coefficient under base cooling condition (W⋅K−1) C̃CCGN base Base cooling CCC normalized for geometry (W⋅K−1⋅m−3) C̃CCHG base Base cooling CCC normalized for heat generation (W⋅K−1⋅Ah−2⋅Ω-1) CCCside Cell cooling coefficient under side cooling condition (W⋅K−1) C̃CCGN side Side cooling CCC normalized for geometry (W⋅K−1⋅m−3) C̃CCHG side Side cooling CCC normalized for heat generation (W⋅K−1⋅Ah−2⋅Ω-1) CR C rate (h−1) hinsu Effective heat transfer coefficient for heat loss through insulation layer (W⋅m−2⋅K−1) L Cell height (m) λlink Effective thermal conductivity between the jellyroll base and the metal can (W⋅m−1⋅K−1) q Cell heat generation rate (W) Q Cell rated capacity (Ah) Qbase Heat rejection rate through cell base (W) Qext Heat extraction rate (W) Qgen Heat generation rate (W) Qside Heat rejection rate through cell side (W) R Cell electrical resistance (Ω) R0 Instantaneous ohmic resistance (Ω) S1 Average temperature integral over time (K⋅s) S2 Temperature difference integral over time (K⋅s) t Time (s) Tavg Cell volume-averaged temperature (K) Tmin Minimum temperature within cell (K) Tmax Maximum temperature within cell (K) Ttarget Target temperature (K) ΔT Temperature difference between max and min temperature within cell (K) ΔTavg Metric to gauge cell overall cooling rate (K) ΔTaxial Temperature difference between the top and bottom of the cell can (K) ΔTgrad Metric to gauge cell emerging thermal gradient (K) ΔTradial Temperature difference between the core and the side surface at the cell height mid-point (K) between temperature and current density, leading to the local temper ature around the tab being higher than elsewhere, thus increasing the volume averaged temperature and the internal thermal gradients [13]. 1. Introduction and motivation demonstrated to predict cylindrical cell electro-thermal performances with relative computational ease and sufficiently high precision. In order to quantify the effectiveness of cooling on the cell, we calculated the cell cooling coefficient (CCC), a previously proposed cell metric designed to quantify the ability to reject heat through different surfaces of a cell [12]. The experimentally measured CCC values of commercial pouch cells were found to lie between 0.004 − 0.06 WK−1Ah−1 for tab cooling [12] and between 0.6 −0.7 WK−1Ah−1 for surface cooling [22], where a larger value of the CCC corresponds to improved heat rejection ability. While surface cooling can extract heat more efficiently than tab cooling, it can also lead to accelerated degra dation [11,23]. The experimentally measured and modelled surface cooling CCC for a prismatic cell was 0.02 −0.06 WK−1Ah−1 [8], a low value interpreted as due to the cell's large form factor, and thus low surface-to-volume ratio. A recent experimental study reported the base cooling CCC is 0.03 WK−1Ah−1 [24] for a commercial 2170 cylindrical cell with single-tab design. There currently is no published value for an experimentally obtained CCC on either continuous tab cylindrical cells, or any design of 4680 cells, presumably because they are still not easily available. i This study is motivated by the advent of cylindrical cells with increasing format factor, and the ensuing need to design and thermally manage them in an efficient manner. It provides two significant con tributions: firstly, it reveals the complete mechanisms through which the ‘tabless design’ acts as an enhancer and enabler of large format cylin drical cells, and secondly, it describes a procedure to identify the optimal thermal management for any future large format cell that may be built. The manuscript is organized as follows: Section 2 presents the model setup of the distributed electro-thermal ECN model, including detailed internal structures. The role of tab as electrical connectors is explored in Section 3. In Section 4, the model is validated against base cooling cell cooling coefficient (CCC) situation. These bespoke experi ments were chosen for their suitability to validate the accuracy of the representation in the model of the thermal connections between the jellyroll and metal. Then the benefit from thermal connections is pre sented in Section 4. 1. Introduction and motivation Therefore, it is challenging to increase the size of cylin drical cells beyond the until now common diameters of 18 mm (1865 cell) and 21 mm (2170 cell), without compromising their ability to reject heat radially. Although end cooling of cylindrical cells accesses a smaller surface area, thus leading to a lower surface-to-volume ratio compared to side cooling, it benefits from the relatively high thermal conductivity of the jellyroll in the axial direction [13]. End cooling also has the advantage of a constant surface-to-volume ratio when cylindrical cell designs with increasing radius (but constant height) are considered. Thermal management is also known to play a key role in the thermal performance of cylindrical cells [19]. Until recently, the majority of three-dimensional cylindrical cell models [20,21] only included the jellyroll, thus ignoring the internal and external structure of the cell, such as metal can, and the effect of internal thermal connections, such as between the jellyroll and the can. These aspects of the cell design should not be ignored whenever modelling the effect of thermal management [13]. It has been shown that conductive side cooling is the most efficient method for a common 2170 cylindrical cell (model: LG M50T), when considering its jellyroll radius and detailed internal structure [13]. For the newer 4680 continuous tab cell [17], the jellyroll is thicker in the radial direction and the internal structure is different from previous The effectiveness of any standard cooling approach is affected by the quality of the thermal connection between the jellyroll and the can. In cylindrical cells the number of tabs between current collectors in the jellyroll and can have usually been optimized for electrical rather than thermal connection. Numerical studies have shown that the number and position of tabs can affect the heat generated by a cell as a by-product of poor electrical design [14,15]. One solution to mitigate the thermal issues in cylindrical cells is through improved tab design [16]. Single-tab (one tab for the positive current collector and one for the negative current collector) and multi- tab designs are common for existing commercial cylindrical cells. The limitation with such tab designs is caused by the positive feedback 2 Applied Energy 349 (2023) 121548 S. Li et al. cylindrical cells. Hence, the most effective cooling strategy for the 4680 cell remains so far unknown. 1. Introduction and motivation In Section 5, the large format cell model is then used as a beginning of life cell design tool, to investigate how to thermally manage the large format cylindrical cells in an effective manner with immediate implication to battery engineering industry. Section 6 con cludes this paper. To quantitatively investigate the effects of both cell design and thermal management, a cell-level distributed model with detailed in ternal structure is needed. There are broadly two categories of cell-level models in literature: continuum models and equivalent circuit models. Derived from the pseudo two-dimensional (P2D) physics-based model [25–28], various Multi-Scale Multi-Dimensional (MSMD) models have been proposed to accelerate the simulation from electrochemical sub- domains up to the cell level [14,21,29–32]. Newman, Tiedemann, Gu and Kwon (NTGK) et al. [33–36] pioneered a semi-empirical model to explore many aspects of cell behavior at the cell scale [37–40]. How ever, these types of models are notoriously difficult to parameterize with confidence [41]. Besides, all aforementioned models do not include the detailed cell internal structure, rather focusing on the amount of active material and interfaces. A recent distributed equivalent circuit network (ECN) model with detailed internal structures [13] has been 3.1. Cell voltage performance Cylindrical cells of two sizes, 2170 and 4680, are modelled using the 3D distributed electro-thermal modelling framework validated in our previous work [13]. The dimensions of the 2170 and 4680 cylindrical cells used in the distributed ECN model are shown in Table 1. The di mensions of the 2170 cell are those of the LG M50T cell. A small mismatch between the reported values of the “real cell” and those in the “model” is caused by imposing the constraint of integer layer number. The ensuing difference in electrode size is similar to errors expected due to electrode and current collector thickness measurements. For a commercially relevant 4680 cell, most required dimensions are not available. A virtual tabless 4680 cell model is built here by assuming that the electrodes' materials and thicknesses are the same as in a LG M50T cell. The rated capacity of the 4680 virtual cell is calculated by scaling the electrode plate area of the 2170 counterpart. As a result, the capacity for the 4680 cell is 5.4 times higher than that of the 2170 cell. The electrical performance of the all-tab and the single-tab designs is studied for both the 2170 and 4680 cells, for a fixed discharge current of 1.5C. This value was chosen as extreme enough to show the effect of tab design, while still within the limits of the LG M50T datasheet. The thermal boundary is in all cases convective cooling on all surfaces with a heat transfer coefficient of 30 Wm−2K−1 and ambient temperature of 25 ◦C. The discharge performance and thermal behavior of single-tab and all-tab designs of a 2170 cylindrical cell are compared in Fig. 2 for the 1.5C discharge with cut-off voltage of 2.7 V. As shown in Fig. 2(a), the discharge voltage of the all-tab design is higher than that of the single- tab design, with an averaged voltage difference of 65.32 mV. This voltage difference is explained by the fact that in the single-tab design there is a longer current pathway through the current collector to the tab and hence the cell has a higher internal resistance. For the same reason, the volume-averaged temperature Tavg for the all-tab design (45.85 ◦C) is lower than that calculated for the single-tab design (49.23◦C), as shown in Fig. 2(b). 3.1. Cell voltage performance The volume-averaged temperature is defined as the weighted average temperatures of all the units in the cell, with the unit thermal mass (or heat capacity) as the weighing factor, thus including the thermally inhomogeneous and anisotropic structure of the cell. The thermal gradient ΔT (i.e. the difference of maximum temperature Tmax and minimum temperature Tmin within the jellyroll domain) for both tab designs is compared in Fig. 2(c). The cell with all-tab design exhibits lower thermal gradients across the jellyroll than the single-tab one. It has been shown that higher thermal gradients contribute to accelerated degradation [11]. The following tab designs for electrical and thermal contact are studied for the 2170 and 4680 cells, in order to identify their effects on the electrical and thermal bottlenecks to cell performance: 1. Single-tab design. A single negative tab is located at the outermost edge of the negative current collector, and a single positive tab is located a third of the distance from the core to the outer end of the positive current collector. Between the jellyroll and the metal can at the cell base and top, there are two main sources of thermal paths - a separator-metal thermal contact and the tabs. For the 2170 cell, this configuration reflects the structure found inside the LG M50T cell during tear-down. Fig. 1(d) shows the tab configuration and separator-metal contact for the negative side. 2. Dual-tab design. The negative current collector has two tabs attached, one placed at each of its end edges. The positive current collector has two tabs attached, one located at a third of the distance from the inner to the outside edge of the positive current collector, and the other tab located at the outermost edge of the current col lector. Separator-metal thermal contact between jellyroll and metal can for negative and positive side. Fig. 3(a) shows the tab configu ration and separator-metal contact for the negative side. In the thermal model, both irreversible and reversible (entropic) heat generation by the electrodes is considered. Irreversible heat generation is considered for current collectors. The total heat generation from a cell is calculated as the sum over all the sub-elements of a cell. The detailed equations and assumptions are listed in the previous modelling work by Li et al. [13]. The total heat generation from current collectors and the electrode/separator/electrode unit is illustrated in Fig. 2(d) during the 1.5C discharge process. 2. Model description A cylindrical cell is composed of a jellyroll (current collector, elec trodes, separator soaked), internal tabs and the outside metal can filled with electrolyte. Fig. 1 shows the schematic description of cylindrical cells with single and continuous tab (or tabless). The cross-section schematic of a single-tab cylindrical cell is shown in Fig. 1(a). For the single-tab design, the negative side of the jellyroll is electrically and thermally connected to the metal can by the single tab, as illustrated in Fig. 1(b). Fig. 1(d) shows the negative side for the disassembled LG M50T cell. As it can be seen, other than this single tab, the remaining space between the jellyroll base and metal can is filled with separator to Fig. 1. Cell diagram for the two cylindrical cell designs. (a) Cross section of cylindrical cell with single-tab design. The geometry details between jellyroll and metal can are zoomed in for (b) single-tab design and (c) tabless (or continuous-tab) design. The negative side for (d) an opened 2170 LG M50T cell, obtained experi mentally, and (e) a 4680 Tesla tabless cell jellyroll [42]. Fig. 1. Cell diagram for the two cylindrical cell designs. (a) Cross section of cylindrical cell with single-tab design. The geometry details between jellyroll and metal can are zoomed in for (b) single-tab design and (c) tabless (or continuous-tab) design. The negative side for (d) an opened 2170 LG M50T cell, obtained experi mentally, and (e) a 4680 Tesla tabless cell jellyroll [42]. 3 S. Li et al. Applied Energy 349 (2023) 121548 jellyroll and the metal can for the negative and the positive sides is formed by the tab and separator-metal contact. Fig. 3(a) shows the tab configuration and separator-metal contact for the negative side. 4. Tabless (continuous-tab) design. The positive and negative current collectors at top/base sides are folded to make plane connections with the plane terminals (metal can top/base sides), as shown in Fig. 1(e). The metal-metal thermal contact between the jellyroll and the can is made. jellyroll and the metal can for the negative and the positive sides is formed by the tab and separator-metal contact. Fig. 3(a) shows the tab configuration and separator-metal contact for the negative side. prevent short-circuit. 2. Model description Since the thermal conductivity of the separator is orders of magnitude lower than that of the metal can and the current collector, this single tab forms the main heat rejection path from the jellyroll base to the outside metal can. i 4. Tabless (continuous-tab) design. The positive and negative current collectors at top/base sides are folded to make plane connections with the plane terminals (metal can top/base sides), as shown in Fig. 1(e). The metal-metal thermal contact between the jellyroll and the can is made. i 4. Tabless (continuous-tab) design. The positive and negative current collectors at top/base sides are folded to make plane connections with the plane terminals (metal can top/base sides), as shown in Fig. 1(e). The metal-metal thermal contact between the jellyroll and the can is made. The continuous tab (or tabless) design is sketched in Fig. 1(c). In comparison to the single-tab design, the addition of a large number of connection points with the can is expected to greatly reduce the bottleneck for both electrical connection and heat rejection via the base (and cap). Fig. 1(e) schematically shows the negative side for the Tesla 4680 cell, from which it can be assumed that the jellyroll base, formed by copper tabs instead of separator, is in direct contact with the can. 3.1. Cell voltage performance The heat generation from the current collectors for the all-tab design is negligible compared with their contribution to heat generation in the single-tab design. For all the usual criteria, such as larger discharge energy, lower average temperature and lower temper ature gradient, the all-tab design performs better than the single-tab design. 3. All-tab design. This design mimics an electrical connection of tabs without the respective thermal connection. The positive and nega tive current collectors are electrically connected to the terminals via one tab for each spiral loop. The thermal contact between the 3. All-tab design. This design mimics an electrical connection of tabs without the respective thermal connection. The positive and nega tive current collectors are electrically connected to the terminals via one tab for each spiral loop. The thermal contact between the Table 1 Features of the model for 2170 and 4680 cell. Features 2170 LG M50T (real cell) 2170 LG M50T (model) 4680 ‘tabless’ cell (model) Cell diameter (mm) 21.00 21.78 46.44 Cell height (mm) 70.00 70.00 80.00 Surface-to-volume ratio (mm−1) 0.22 0.22 0.11 Electrode length (mm) 915 884 4183 Electrode plate area (mm2) 1.22e5 1.17e5 6.33e5 Tab Length/Width/Thickness (mm) 11/3.5/ 0.165 11/3.5/ 0.165 N/A Rated capacity (Ah) 5.00 5.00 27.07 g A similar set of simulation is performed for 4680 cylindrical cells with single-tab, dual-tab and all-tab designs. The schematics for those tab designs are illustrated in Fig. 3(a). At first the discharge capacity of single-tab design under different C rates is checked in Fig. 3(b). The discharge capacity under 0.1C discharge (pseudo OCV test), is close to the rated capacity (27.07 Ah). The discharge capacity is significantly reduced as the C rate increases from 0.1C to 1.5C. The terminal voltage of this 4680 cell for 1.5C falls below the cutoff voltage of 2.7 V from the start of discharge (Fig. 3(b)) unlike the 2170 single-tab cell (Fig. 2(a)). This effect can be explained by considering the length of the jellyroll in each of the cells. Fig. 3(c) shows the schematic representation of the unwound current collector for the 2170 (single-tab) cell and the 4680 (all three cases of tab designs) cells. When a single-tab design is applied, Table 1 Features of the model for 2170 and 4680 cell. 3.2. Power performance In this section, the pulse power capability for the four cells (2170 single-tab, 2170 all-tab, 4680 dual-tab and 4680 all-tab cells) is inves tigated, as it is a key figure of merit in applications. The power capability is defined as the maximum discharge power that can be sustained for 10 s while also maintaining the terminal voltage above a cutoff voltage of 3.0 V. Fig. 4(a) shows the power capability when starting from different SoC between 100% and 10%. For all cells, the power capability de creases monotonically with a decrease in starting SoC. The 4680 cell with all-tab design provides the highest power among the four cells for all starting SoC levels. At 100% SoC, the power capability of the 4680 all-tab cell is 543.7 W (corresponding to 6.7C), while the power capa bility of the 2170 single-tab cell, i.e., the LG M50T cell, is 78.4 W (corresponding to 5.2C). The power capability of the 4680 all-tab cell is 6.9 times that of the 2170 single-tab cell, i.e. the LG M50T cell. The power capability of a 4680 cell is halved when the all-tab design is replaced with dual-tab design, as shown in Fig. 4(a). Dual-tab design causes the lower cutoff voltage to be reached sooner than in the case of all-tab design. The power capacity for 2170 cell single-tab and all-tab i The all-tab design does not exhibit the capacity limitation for 1.5C discharge, reaching close to full rated capacity as shown in Fig. 3(d). As an intermediate solution, a dual-tab design is also considered. While the discharge capacity of the dual-tab design only approaches 80% of the rated capacity, it performs markedly better than the single-tab design, showing that even adding an extra tab of each polarity can greatly improve performance by reducing internal resistance. These results predict that the electrical performance of the 4680 cell is extremely sensitive to the number of tabs. As illustrated in Fig. 3(c), the long electrical path for the 4680 cell is effectively reduced by increasing the number of tabs (i.e. dual-tab design), with the all-tab design minimizing the electrical path. Unlike for the 2170 cell, the 4680 cell must have a dual-tab or all-tab design to be viable. The total heat generation throughout the discharge from current collectors and electrodes for dual-tab and all-tab designs is compared in Fig. 3(e). Table 1 F (a) 0 1 2 3 4 5 2.7 3.0 3.3 3.6 3.9 4.2 4.5 ) V ( e g a tlo V Discharge capacity (Ah) 2170 single-tab 2170 all-tab (a) (b) 0 1 2 3 4 5 2.7 3.0 3.3 3.6 3.9 4.2 4.5 ) V ( e g a tlo V Discharge capacity (Ah) 2170 single-tab 2170 all-tab 0 1 2 3 4 5 25 35 45 55 65 e r u t a r e p m e t e g a r e v A Tavg ) C °( Discharge capacity (Ah) 2170 single-tab 2170 all-tab (b) 0 1 2 3 4 5 25 35 45 55 65 e r u t a r e p m e t e g a r e v A Tavg ) C °( Discharge capacity (Ah) 2170 single-tab 2170 all-tab (a) (b) Discharge capacity (Ah) (c) Discharge capacity (Ah) 0 1 2 3 4 5 0 3 6 9 12 15 e c n e r e ffid e r u t a r e p m e T ∆T = Tmax-Tmin ) C °( Discharge capacity (Ah) 2170 single-tab 2170 all-tab (c) (d) (d) 2170 single-tab 2170 all-tab 0 3000 6000 9000 12000 ) J ( n oit a r e n e g t a e H Aluminum foil Copper foil Electrodes Fig. 2. Comparison of single-tab and all-tab designs for a 2170 cylindrical cell during 1.5C discharge under convective thermal boundary condition: (a) terminal voltage, (b) volume-averaged jellyroll temperature, (c) maximum temperature difference across the jellyroll and (d) total heat generation of electrodes and current collectors during discharge. major source of heat generation in the dual-tab 4680, a role they did not play for the 2170 cell (as shown in Fig. 2(d)). the electrical path (indicated by red arrows in Fig. 3(c)) is significantly longer for the 4680 cell than for the 2170 cell, as the current collector lengths are 4.18 m vs 0.88 m, as given for the electrode lengths in Table 1. Features of the model for 2170 and 4680 cell. The voltage drop at the start of the discharge is hence significant for the single-table 4680, due to its significantly higher resistance. Table 1 F Features of the model for 2170 and 4680 cell. 4 Applied Energy 349 (2023) 121548 S. Li et al. (a) (b) (c) (d) 0 1 2 3 4 5 2.7 3.0 3.3 3.6 3.9 4.2 4.5 ) V ( e g a tlo V Discharge capacity (Ah) 2170 single-tab 2170 all-tab 0 1 2 3 4 5 25 35 45 55 65 e r u t a r e p m e t e g a r e v A Tavg ) C °( Discharge capacity (Ah) 2170 single-tab 2170 all-tab 0 1 2 3 4 5 0 3 6 9 12 15 e c n e r e ffid e r u t a r e p m e T ∆T = Tmax-Tmin ) C °( Discharge capacity (Ah) 2170 single-tab 2170 all-tab 2170 single-tab 2170 all-tab 0 3000 6000 9000 12000 ) J ( n oit a r e n e g t a e H Aluminum foil Copper foil Electrodes Fig. 2. Comparison of single-tab and all-tab designs for a 2170 cylindrical cell during 1.5C discharge under convective thermal boundary condition: (a) terminal voltage, (b) volume-averaged jellyroll temperature, (c) maximum temperature difference across the jellyroll and (d) total heat generation of electrodes and current collectors during discharge. 3.2. Power performance As the single-tab configuration cannot be discharged at 1.5C, it is not considered here. The current collectors can be seen to act as a 5 5 S. Li et al. Applied Energy 349 (2023) 121548 compared in Fig. 4(b) for the appropriate y-axis power range. design improves the power for the 2170 cell, but not nearly i 4. Benefit from thermal connection mparison of tab designs for a 4680 cylindrical cell during 1.5C discharge under convective thermal boundary condition. (a) Schematic of single-tab, dua ab designs (legend as in Fig. 1). (b) Capacity check for 4680 single-tab cell under different C rates. (c) Schematic of the electrical path through the curre single-tab 2170 and 4680 cell, dual-tab and all-tab 4680 cell. (d) Terminal voltage for single-tab, dual-tab and all-tab design. (e) Total heat generation nd current collectors for dual-tab and all-tab design during discharge. Fig. 3. Comparison of tab designs for a 4680 cylindrical cell during 1.5C discharge under convective thermal boundary condition. (a) Schematic of single-tab, dual- tab and all-tab designs (legend as in Fig. 1). (b) Capacity check for 4680 single-tab cell under different C rates. (c) Schematic of the electrical path through the current collector for single-tab 2170 and 4680 cell, dual-tab and all-tab 4680 cell. (d) Terminal voltage for single-tab, dual-tab and all-tab design. (e) Total heat generation of electrodes and current collectors for dual-tab and all-tab design during discharge. 4. Benefit from thermal connection designs is compared in Fig. 4(b) for the appropriate y-axis power range. The all-tab design improves the power for the 2170 cell, but not nearly as significantly as for the 4680 cell (all-tab vs dual-tab design), as the electrical path for a 4680 cell is significantly longer than that for the 2170 cell. The maximum C rate is shown in Fig. 4(a) for the four cells. The maximum C rate for the all-tab design is significantly higher than its single-tab/dual-tab counterpart, for both 4680 and 2170 cells. For the same cut-off voltage, the cell with all-tab design allows higher C rate capability in comparison to single-tab/dual-tab design. The C rate capability for all-tab 2170 and all-tab 4680 cell is similar, because the two cells have similar structure in the case where the extra resistances induced by tab configuration are minimized. 4.1. Model validation for the prediction of the base cooling Cell Cooling Coefficient (CCC) on 2170 cells An obvious feature of the tabless design is that the contact between the jellyroll and the can at the negative side is made via the copper tabs, instead of via separator layers, with thermal conductivity of 398 Wm−1 K−1 vs. 0.34 Wm−1 K−1 [43]. The metal-metal thermal connection makes heat dissipation much more efficient than for a separator-metal connection. The Cell Cooling Coefficient (CCC) has shown to be a meaningful metric to quantify the heat generation and rejection ability of a battery cell [22]. In this section, the CCC is calculated for the various cells based on virtual experiments run on the 3D distributed ECN model, 6 Applied Energy 349 (2023) 121548 S. Li et al. Fig. 4. Power capability for the four cylindrical cells when discharged from different SoC. (a) Maximum capacity and C rate for the 2170 single-tab and all-tab cells, and the 4680 dual-tab and all-tab cells. (b) Zoomed-in maximum power for the 2170 single-tab cell and the 2170 all-tab cell. Fig. 4. Power capability for the four cylindrical cells when discharged from different SoC. (a) Maximum capacity and C ra and the 4680 dual-tab and all-tab cells. (b) Zoomed-in maximum power for the 2170 single-tab cell and the 2170 all-tab ylindrical cells when discharged from different SoC. (a) Maximum capacity and C rate for the 2170 single-tab and all-tab cells ls. (b) Zoomed-in maximum power for the 2170 single-tab cell and the 2170 all-tab cell. Fig. 4. Power capability for the four cylindrical cells when discharged from different SoC. (a) Maximum capacity and C rate for the 2170 single-tab and all-tab cells, and the 4680 dual-tab and all-tab cells. (b) Zoomed-in maximum power for the 2170 single-tab cell and the 2170 all-tab cell. and used to compare the heat rejection ability associated with the different tab designs. restrict all thermal pathways except the one of interest: base cooling path. The cooling path is designed to develop an easily measurable temperature gradient along its length when heat flows from the cell. The experimental setup involves connecting the cell at the top and base with g The experiments are set up on a base cooled LG M50T for the 2170 cell, yielding a value for CCCbase. 4.1. Model validation for the prediction of the base cooling Cell Cooling Coefficient (CCC) on 2170 cells For the model setup, the same pulsing current is applied as in the experiment, as shown in the inset of Fig. 5(a). The base surface is set as the cooling area. The thermal boundary condition for the side surface and the positive cap surface is assumed to be characterized by a small heat transfer coefficient hinsu to account for the small heat loss through the insulation layer surrounding those surfaces and down the tabs con necting the cell electrically. The effective thermal conductivity between the jellyroll base and the metal can, λlink, is set as a small value in the model to represent the weak heat transfer through one tab and separator layers. The values of hinsu = 3.5 Wm−2 K−1 and λlink = 2.6 Wm−1 K−1 are found to allow the model predicted temperature and heat transfer rate to match the measured values. In the CCC simulation test, pulses of 1.5C discharge and 1.5C charge are applied to three cells: 2170 single-tab (LG M50T cell as reference, separator-metal connection between jellyroll and can), 4680 all-tab (separator-metal thermal connection) and 4680 tabless (metal-metal thermal connection). To account for the effects of cell size, a geometry- normalized CCC value is calculated, as introduced by Marzook et al. for cylindrical cells [24]. The nomalized C̃CCGN base [W K−1 m−3] is calculated from the temperature gradient across the height of the cell ΔTaxial/L and the base cooling rate per area of the base Qbase/Abase: C̃CCGN base = Qbase/Abase ΔTaxial/L = CCCbase Abase/L , (2) C̃CCGN base = Qbase/Abase ΔTaxial/L = CCCbase Abase/L , (2) Under these conditions, most of the extracted heat is transferred through the cell base. The heat extraction rate Qext and the heat gener ation rate Qgen predicted by the model are shown in Fig. 5(a). Initially, the heat generation rate decreases and the heat extraction rate increases. This behavior is caused by the increasing cell temperature. The resis tance decreases with temperature for the LG M50T cell [13], thus lowering the heat generation rate Qgen, which is governed by irreversible losses. The heat extraction rate Qext, on the other hand, is proportional to the difference between the internal cell temperature at the cell base and the external cooling temperature of the cooling plate. This difference increases as the internal cell temperature increases. 4.1. Model validation for the prediction of the base cooling Cell Cooling Coefficient (CCC) on 2170 cells spot welded nickel tabs clamped between brass blocks. A tab is spot welded as closely to the edge of the base as possible in order to minimize its effect on the cooling path. The connected cell is encased in insulating foam with only the base of the cell in contact with a Peltier controlled cooling fin. Two main assumptions have been made in calculating the CCCbasefrom the experimental data: 1) that the main heat rejection pathway lies along the cell axial direction and all heat is rejected through the base of the cell, and 2) that the heat generation is all caused by the cell itself, thus ignoring heat generation from the tab weld. More details on the experimental setup can be found in Marzook et al. [24], done on the same cell type LG M50T. In the experiment, a switching discharge/charge current with the frequency of 1 Hz is applied until the cell and rig thermalize. These highly specific conditions are replicated under different discharge/charge rates (0.25C, 0.5C, 0.75C. 1C, 1.25C, 1.5C, 1.75C and 2C), different temperature (20 ◦C and 30 ◦C) and initial SoC (50% and 75%). configuration for the tabless cell, shown in Fig. 1(e), is expected to improve the rate of heat rejection through the cell base. The benefit of improved thermal connection by the tabless connection is quantified by comparing the different options for internal thermal connection be tween the jellyroll base and the metal can base. For the all-tab scenario, the thermal connection between jellyroll base and metal can base at the negative side of the cell is set to be the same as that found in the 2170 LG M50T cell, i.e., the same value is assumed for the effective thermal conductivity λlink for the connection between jellyroll base and metal can. For the tabless scenario, it is assumed that the copper foil of the tabs and the metal can are in perfect thermal contact with each other at the negative terminal. The other thermal connections, such as that between the positive current collector and the can, as well as that between the electrodes and the can, remain unchanged from the separator-can thermal contact scenario, i.e., with thermal conductivity λlink = 2.6 Wm−1K−1. An equivalent setup is simulated on the positive connection, where the aluminum foil and the metal can are assumed to be in perfect thermal contact. 4.1. Model validation for the prediction of the base cooling Cell Cooling Coefficient (CCC) on 2170 cells The experimental rig is designed to 7 0 5 10 15 20 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Qbase ) W ( Taxial (°C) (a) (b) (c) (d) 25.10 ºC 29.93 ºC Green: SoC=50%, Tamb=20 ºC Red: SoC=50%, Tamb=30 ºC Blue: SoC=75%, Tamb=20 ºC 3.75 A 5.0 A 6.25 A 7.5 A 8.75 A 10.0 A Exp Sim Exp fitting Sim fitting 0 10 20 30 40 50 60 70 25 27 29 31 33 ) C °( e r u t a r e p m e T Distance from base (mm) exp sim Fig. 5. Model validation results for the CCC experiment on 2170 cylindrical LG M50T cell. (a) Model predicted heat rate evolution for a 1C (5A) current during th virtual CCC experiment. (b) Predicted temperature distribution throughout the cell at steady state for a 1C (5A) pulsed current. (c) Simulation and experimental da for temperature at different side surface locations along the axial direction of the cell can. (d) Simulation and experimental data for the base cooling rate Qbase vs th temperature difference between positive and negative side ΔTaxial under different current pulse amplitude, cell SoC and ambient temperature. (a) (b) (b) (a) 25.10 ºC 29.93 ºC (c) ( 0 10 20 30 40 50 60 70 25 27 29 31 33 ) C °( e r u t a r e p m e T Distance from base (mm) exp sim 0 5 10 15 20 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Qbase ) W ( Taxial (°C) (d) Green: SoC=50%, Tamb=20 ºC Red: SoC=50%, Tamb=30 ºC Blue: SoC=75%, Tamb=20 ºC 3.75 A 5.0 A 6.25 A 7.5 A 8.75 A 10.0 A Exp Sim Exp fitting Sim fitting (d) (c) Fig. 5. Model validation results for the CCC experiment on 2170 cylindrical LG M50T cell. (a) Model predicted heat rate evolution for a 1C (5A) current during the virtual CCC experiment. (b) Predicted temperature distribution throughout the cell at steady state for a 1C (5A) pulsed current. (c) Simulation and experimental data for temperature at different side surface locations along the axial direction of the cell can. (d) Simulation and experimental data for the base cooling rate Qbase vs the temperature difference between positive and negative side ΔTaxial under different current pulse amplitude, cell SoC and ambient temperature. 7 Applied Energy 349 (2023) 121548 S. Li et al. 4.1. Model validation for the prediction of the base cooling Cell Cooling Coefficient (CCC) on 2170 cells Qbase is a main contributor to Qext, alongside the heat flow rate through the side and top surfaces of the can. The experimental and simulation results for the temperature difference ΔTaxial and the base cooling rate Qbase are in good agreement, as shown in Fig. 5(d) for different pulsing currents, SoCs and ambient temperature conditions. Under these pulsing currents, the CCC is around 0.14 W K−1 for both model prediction and experimental measurement. where Qbase [W] is the heat rejection rate through the cell base [22]. Qbase is a main contributor to Qext, alongside the heat flow rate through the side and top surfaces of the can. The experimental and simulation results for the temperature difference ΔTaxial and the base cooling rate Qbase are in good agreement, as shown in Fig. 5(d) for different pulsing currents, SoCs and ambient temperature conditions. Under these pulsing currents, the CCC is around 0.14 W K−1 for both model prediction and experimental measurement. C̃CCHG base = CCCbase Q2⋅R0 , (3) C̃CCHG base = CCCbase Q2⋅R0 , (3) where R0 [Ω] is the instantaneous ohmic resistance obtained indepen dently from the simulated instantaneous voltage drop under an applied current of 1.5C at 25 ◦C from 50% SoC. The value of R0 for the 2170 single-tab cell, the 4680 all-tab cell and the 4680 tabless cell are found to be 3.71 × 10−2 Ω, 5.14 × 10−3 Ω and 4.88 × 10−3 Ω, respectively. With these values, the C̃CCHG base for 4680 tabless cell is the highest among these three cells (almost twice of the non-tabless 2170 single-tab and 4680 all- where R0 [Ω] is the instantaneous ohmic resistance obtained indepen dently from the simulated instantaneous voltage drop under an applied current of 1.5C at 25 ◦C from 50% SoC. The value of R0 for the 2170 single-tab cell, the 4680 all-tab cell and the 4680 tabless cell are found to be 3.71 × 10−2 Ω, 5.14 × 10−3 Ω and 4.88 × 10−3 Ω, respectively. With 4.1. Model validation for the prediction of the base cooling Cell Cooling Coefficient (CCC) on 2170 cells Around 3500 s, the heat extraction rate Qext and the heat generation rate Qgen balance, as the system reaches steady state and the CCCbase can be calculated. The model-predicted temperature distribution inside the cell during steady state reached for a 1C (5 A) current is shown in Fig. 5(b). The thermal gradient forms mostly along the axial direction, reaching a temperature difference ΔTaxial = 4.83◦C between the top and bottom of the cell can. The measured and predicted temperatures at steady state along the axial direction are compared in Fig. 5(c). The base cooling CCCbase [W K−1] of this LG M50T cell is given as: where L [m] is the cell height and Abase [m2] is the cell base area. The predicted temperature gradient ΔTaxial/L and base cooling rate per area Qbase/Abase at steady state for the three cells are compared in Fig. 6(a). The full parameters of the model is listed in Table 2. The base cooling heat extraction rates Qbase/Abase are similar for the three cells, while the thermal gradients vary significantly. The 4680 tabless cell experiences nearly half the axial thermal gradient of the 2170 and 4680 all-tab cells. The metal-metal contact enables efficient heat transfer in the tabless cell, while the separator-metal contact in the single tab and all tab cells forms a thermal bottleneck. The CCC results are shown in Fig. 6(b). The normalized C̃CCGN base for 4680 all-tab cell is similar to that of the 2170 cell. The tabless design in the 4680 cell yields the highest C̃CCGN base among the three cells, by a factor of two. The value of CCCbase (i.e. before considering the effect of cell geometry) for the 4680 tabless cell is 1.15 WK−1. This value is higher than the value corresponding to traditionally better cooling scenarios, such as surface-cooled pouch cells, that have been found to have CCC values ranging within 0.99 WK−1 for a commercial pouch cell [22]. CCCbase = Qbase ΔTaxial , (1) CCCbase = Qbase ΔTaxial , (1) An alternative comparison can be made by normalizing CCC with respect to the heat generation rate, q = (CR⋅Q)2⋅R, where CR is the C rate, Q [Ah] is the cell rated capacity and R [Ω] is the cell resistance. The CCC normalized for heat generation C̃CCHG base [W K−1 Ah−2 Ω−1] can be approximated as: where Qbase [W] is the heat rejection rate through the cell base [22]. Table 2 Table 2 Parameters of the model for base cooling and side cooling CCC. Table 2 Parameters of the model for base cooling and side cooling CCC. 2170 single-tab 4680 all-tab 4680 tabless Abase (mm2) 346.36 1661.90 1661.90 Aside (mm2) 4618.14 11,561.05 11,561.05 L (mm) 70 80 80 D (mm) 21 46 46 ΔTaxial (◦C) 11.12 13.30 6.46 Base Qgen (W) 2.06 8.36 8.01 Base Qext (W) 2.06 8.36 8.01 Qbase (W) 1.60 7.35 7.38 ΔTradial (◦C) 2.15 7.21 6.71 Side Qgen (W) 2.19 8.83 8.42 Side Qext (W) 2.19 8.83 8.42 Qside (W) 2.19 8.79 8.38 arameters of the model for base cooling and side cooling CCC. C̃CCGN side = Qside/Aside ΔTradial/0.5D = CCCside Aside/0.5D, (5) (5) here Aside [m2] is the surface area of the side of the cell. The CCC normalized for heat generation C̃CCHG base is: here Aside [m2] is the surface area of the side of the cell. The CCC normalized for heat generation C̃CCHG base is: C̃CCHG side = CCCside Q2⋅R0 , (6) C̃CCHG side = CCCside Q2⋅R0 , (6) tab cells), showing superior thermal performance, as shown in Fig. 6(b). Some applications employ partial surface cooling as their thermal management approach. For this reason, the analysis above is repeated for three cylindrical cells under surface cooling. In this reference case, the entire surface is cooled. The side cooling CCCside [W K−1] is defined as: where R0 is calculated at 50% SoC under 25 ◦C initial temperatures. The predicted temperature gradient ΔTradial/0.5D and the side cooling rate per area Qside/Aside at steady state for the three cells are compared in Fig. 6(c). The full parameters of the model are listed in Table 2. The thermal gradient along the radial direction for the 4680 cell is higher than that of 2170 single-tab cell, because of the larger radial dimension, and thus longer heat path, along an axis with relatively poor thermal conductivity. The side cooling CCC results are shown in Fig. 6(d). The normalized C̃CCGN side and C̃CCHG side for the 4680 cell (all-tab and tabless) are significantly lower than those of the 2170 cell, in stark contrast to the where R0 is calculated at 50% SoC under 25 ◦C initial temperatures. The predicted temperature gradient ΔTradial/0.5D and the side cooling rate per area Qside/Aside at steady state for the three cells are compared in Fig. 6(c). 4.2. Predicted base and side Cell Cooling Coefficient (CCC) for 4680 cells The cylindrical cell model validated for CCCbaseof a 2170 cell is used to predict the ability of the 4680 cell to be cooled, by calculating base cooling CCCbase and side cooling CCCside. The metal-metal thermal these values, the C̃CCHG base for 4680 tabless cell is the highest among these three cells (almost twice of the non-tabless 2170 single-tab and 4680 all- 8 Applied Energy 349 (2023) 121548 S. Li et al. Fig. 6. Simulated base and side cooling CCC results for 2170 single-tab, 4680 all-tab and 4680 tabless cells. (a) Cell temperature gradient along the height of the cell and heat rejection rate per area from the cell base. (b) Base cooling CCCbase and normalized C̃CCGN base and C̃CCHG base. (c) Cell temperature gradient along the radial direction and the heat rejection rate per area from the cell side surface. (d) Side cooling CCCside and normalized C̃CCGN side and C̃CCHG side. Fig. 6. Simulated base and side cooling CCC results for 2170 single-tab, 4680 all-tab and 4680 tabless cells. (a) Cell temperature gradient along the height of the cell and heat rejection rate per area from the cell base. (b) Base cooling CCCbase and normalized C̃CCGN base and C̃CCHG base. (c) Cell temperature gradient along the radial direction and the heat rejection rate per area from the cell side surface. (d) Side cooling CCCside and normalized C̃CCGN side and C̃CCHG side. surface at the cell height mid-point. This definition is analogous to that of surface cooling for pouch cells, and corresponds to a theoretical measure, enabled by the model, of a cylindrical cell's ability to be cooled through its side surface. Of course, in experiments the core temperature is not easily available. The side cooling normalized for geometry C̃CCGN side [W K−1 m−3] is calculated from the temperature gradient ΔTradial/0.5D and the base cooling rate per area Qbase/Abase, where D [m] is the cell diameter: 4.3. Average temperature and thermal gradients predicted for 4680 cells under normal discharge condition Beyond CCC, the viability of cells can be quantitatively analyzed based on the average temperature and temperature gradients they are expected to experience during operation [13]. The effect of the thermal connection on the transient temperature performance (volume-averaged temperature and thermal gradients) of the 4680 cell is investigated for a 1.5C discharge base cooled by a cooling plate at 25 ◦C and with side surfaces thermally insulated. Table 2 The full parameters of the model are listed in Table 2. The thermal gradient along the radial direction for the 4680 cell is higher than that of 2170 single-tab cell, because of the larger radial dimension, and thus longer heat path, along an axis with relatively poor thermal conductivity. The side cooling CCC results are shown in Fig. 6(d). The normalized C̃CCGN side and C̃CCHG side for the 4680 cell (all-tab and tabless) are significantly lower than those of the 2170 cell, in stark contrast to the CCCside = Qside ΔTradial , (4) (4) where Qside [W] is the heat rejection rate through the cell side surface, and ΔTradial is the temperature difference between the core and the side 9 Applied Energy 349 (2023) 121548 S. Li et al. base cooling results in Fig. 6(b). The side cooling surface-to-volume ratio is inversely proportional to the cell radius, while base cooling surface-to-volume ratio is independent of radius. Therefore, for side cooling, the tabless design does not improve the heat rejection, while the drawback of the larger radius impacts the heat rejection from the core of the 4680 cell. temperature difference between the jellyroll and the metal can at the negative side is significant for the all-tab cell, indicating a thermal bottleneck created by the poor thermal connection pathway, unlike in the tabless cell. i Since base cooling has been shown to be very efficient for a tabless cell, the potential gains that can be achieved by top and base cooling are explored. As shown in Fig. 7, the average cell temperature and the temperature difference can be further decreased by cooling both top and base, in comparison to base-only cooling. However, there are obvious practical hindrances to cooling the top due to the presence of electrical connections and venting points. 4.3. Average temperature and thermal gradients predicted for 4680 cells under normal discharge condition 4.3. Average temperature and thermal gradients predicted for 4680 cells under normal discharge condition 4.4. Choosing the best thermal management for the 4680 tabless cell Using the model developed, two common cooling schemes, top/base cooling and side cooling, are applied on a 4680 tabless cell, in order to evaluate their effect on its thermal performance. All other surfaces are assumed insulated, to somewhat mimic the conditions within a pack, when the cell is surrounded by other heat-generating cells. Convective boundary conditions are used to retrieve the effect of forced air cooling, while conductive boundary conditions are used to retrieve the effect of contact plate cooling. In convective cooling, a heat transfer coefficient of 30 Wm−2 K−1 and an ambient temperature of 25 ◦C are assumed. In conductive cooling, the temperature on the cooling surface is fixed at 25 ◦C. The volume-averaged temperature for the 4680 all-tab cell and tabless cell are shown in Fig. 7(a). For the 4680 all-tab cell, the volume- averaged temperature rise is the highest, reaching 48 ◦C at the end of discharge. In contrast, the cell with tabless design has a significantly lower volume-averaged temperature, around 35 ◦C at the end of discharge. It is expected that a cell with a lower average temperature will degrade slower. The presence and magnitude of thermal gradients is also expected to be strongly correlated to reduced performance and lifetime. The temperature difference ΔT (i.e. the difference between maximum Tmax and minimum temperature Tmin anywhere within the cell) is shown in Fig. 7(b). The thermal gradient for the all-tab design is twice the value of tabless design, indicating that the all-tab cell does not benefit from base cooling as much as the tabless cell. The temperature distribution at the end of discharge is shown in Fig. 7(c). The The simulated volume-averaged temperature for the four cooling schemes considered is shown in Fig. 8(a). The conductive cooling sce narios lead to significantly lower volume-averaged cell temperature than the convection scenarios, due to the higher heat transfer rate be tween the cell and its ambient enabled by the metal-metal conduction than the metal-air convection boundary condition. However, side Fig. 7. Thermal performance for 4680 all-tab and tabless cells during a 1.5C discharge for base cooling and top & base cooling with a plate temperature of 25 ◦C and side surfaces insulated. (a) Volume-averaged temperature and (b) temperature difference across the cell. (c) Temperature distribution at end of discharge (after 2400 s of discharge time). Fig. 7. 4.4. Choosing the best thermal management for the 4680 tabless cell Thermal performance for 4680 all-tab and tabless cells during a 1.5C discharge for base cooling and top & base cooling with a plate temperature of 25 ◦C and side surfaces insulated. (a) Volume-averaged temperature and (b) temperature difference across the cell. (c) Temperature distribution at end of discharge (after 2400 s of discharge time). Fig. 7. Thermal performance for 4680 all-tab and tabless cells during a 1.5C discharge for base cooling and top & base cooling with a plate temperature of 25 ◦C and side surfaces insulated. (a) Volume-averaged temperature and (b) temperature difference across the cell. (c) Temperature distribution at end of discharge (after 2400 s of discharge time). 10 Applied Energy 349 (2023) 121548 S. Li et al. Side conv Side cond Top/base conv Top/base cond Tavg= 71.38 ºC Tavg= 36.32 ºC Tavg= 95.61 ºC Tavg= 29.74 ºC (c) 8.87 ºC -11.33 ºC T(x, y, z) - Tavg 0 5 10 15 20 25 30 25 40 55 70 85 100 ) C °( e r u t a r e p m e t e g a r e v A Discharge capacity (Ah) Side conv Side cond Top/Base conv Top/Base cond 0 5 10 15 20 25 30 0 5 10 15 20 25 ) C °( e c n e r e ffid e r u t a r e p m e T Discharge capacity (Ah) Side conv Side cond Top/Base conv Top/Base cond (a) (b) g. 8. Model predictions of the thermal performance of a 4680 tabless cell during a 1.5C discharge under four thermal management conditions: side convection, side nduction, top/base convection and top/base conduction. (a) Volume-averaged temperature and (b) the maximum temperature difference within the cell. (c) ternal temperature distribution at the end of discharge. 4.4. Choosing the best thermal management for the 4680 tabless cell measures the ability of the cell to maintain the cell internal average temperature close to a target value Ttarget, time-averaged over the discharge process. ΔTavg is given by: conductive cooling causes the largest temperature difference across the cell, as shown in Fig. 8(b). This high level of temperature difference is guaranteed to cause accelerated degradation [11]. Although the tem perature difference for top/base convection is the lowest, this may not the best cooling choice for long cycle life, since the volume-averaged temperature is relatively high, as shown in Fig. 8(a). The temperature distribution for the 4680 cell under the four cooling scenarios at the end of discharge is shown in Fig. 8(c). The thermal gradient within the cell forms along the radial direction for side cooling and mainly along the axial direction for top/base cooling scenarios, for both convection and conduction. Considering the smaller average temperature and thermal gradient, it can be concluded that top/base conductive cooling is the best cooling scheme for the 4680 tabless cell. In contrast, the best cooling approach for the 2170 cell was found to be side cooling [13], due to the larger cooling area of the side surface than the top/base surface. conductive cooling causes the largest temperature difference across the cell, as shown in Fig. 8(b). This high level of temperature difference is guaranteed to cause accelerated degradation [11]. Although the tem perature difference for top/base convection is the lowest, this may not the best cooling choice for long cycle life, since the volume-averaged temperature is relatively high, as shown in Fig. 8(a). The temperature distribution for the 4680 cell under the four cooling scenarios at the end of discharge is shown in Fig. 8(c). The thermal gradient within the cell forms along the radial direction for side cooling and mainly along the axial direction for top/base cooling scenarios, for both convection and conduction. Considering the smaller average temperature and thermal gradient, it can be concluded that top/base conductive cooling is the best cooling scheme for the 4680 tabless cell. In contrast, the best cooling approach for the 2170 cell was found to be side cooling [13], due to the larger cooling area of the side surface than the top/base surface. ΔTavg = S1 t = 1 t ∫t 0 ( Tavg(t) −Ttarget ) ⋅dt, (7) (7) where S1 is the integral of the temperature over time t. 4.4. Choosing the best thermal management for the 4680 tabless cell Ttarget is set here as 25 ◦C, to match the ambient and cooling plate temperature. The second metric ΔTgrad measures the emerging thermal gradient within the cell, time-averaged over the discharge process. ΔTgrad is given as: ΔTgrad = S2 t = 1 t ∫t 0 (Tmax(t) −Tmin(t) )⋅dt, (8) (8) where S2 is the integral of the temperature difference over time t. Tmax and Tmin are the maximum and minimum temperatures in the jellyroll, respectively. For better thermal performance, a cell has as small a value as possible for both metrics. 4.4. Choosing the best thermal management for the 4680 tabless cell 0 5 10 15 20 25 30 25 40 55 70 85 100 ) C °( e r u t a r e p m e t e g a r e v A Discharge capacity (Ah) Side conv Side cond Top/Base conv Top/Base cond 0 5 10 15 20 25 30 0 5 10 15 20 25 ) C °( e c n e r e ffid e r u t a r e p m e T Discharge capacity (Ah) Side conv Side cond Top/Base conv Top/Base cond (a) (b) 0 5 10 15 20 25 30 25 40 55 70 85 100 ) C °( e r u t a r e p m e t e g a r e v A Discharge capacity (Ah) Side conv Side cond Top/Base conv Top/Base cond (a) 0 5 10 15 20 25 30 0 5 10 15 20 25 ) C °( e c n e r e ffid e r u t a r e p m e T Discharge capacity (Ah) Side conv Side cond Top/Base conv Top/Base cond (b) ) (b) (a) Discharge capacity (Ah) Discharge capacity (Ah) Side conv Tavg= 71.38 ºC (c) Side conv Side cond Top/base conv Top/base cond Tavg= 71.38 ºC Tavg= 36.32 ºC Tavg= 95.61 ºC Tavg= 29.74 ºC (c) 8.87 ºC -11.33 ºC T(x, y, z) - Tavg Fig. 8. Model predictions of the thermal performance of a 4680 tabless cell during a 1.5C discharge under four thermal management conditions: side convection, side conduction, top/base convection and top/base conduction. (a) Volume-averaged temperature and (b) the maximum temperature difference within the cell. (c) Internal temperature distribution at the end of discharge. Side conv Side cond Tavg= 71.38 ºC Tavg= 36.32 ºC (c) (c) Side cond Tavg= 36.32 ºC Top/base cond Tavg= 29.74 ºC 8.87 ºC -11.33 ºC T(x, y, z) - Tavg Top/base conv Tavg= 95.61 ºC Tavg= 95.61 ºC Tavg= 29.74 ºC Tavg= 71.38 ºC Tavg= 36.32 ºC Fig. 8. Model predictions of the thermal performance of a 4680 tabless cell during a 1.5C discharge under four thermal management conditions: side convection, side conduction, top/base convection and top/base conduction. (a) Volume-averaged temperature and (b) the maximum temperature difference within the cell. (c) Internal temperature distribution at the end of discharge. 5. Discussion The thermal performances of the 4680 tabless cell and the 2170 single-tab cell are analyzed in this section based on the virtual model experiments run so far. In comparison to the 2170 cell, the 4680 cell is disadvantaged by geometry: 1) the 4680 cell has lower surface-to- volume ratio for side cooling, and 2) the longer radius of 4680 cell can lead to higher radial thermal gradients. In both cases, a tabless design has been demonstrated to improve the thermal performance. Therefore, a comparison between the 2170 cell and 4680 cell is not straightforward and must be conducted separately for each of the different cooling scenarios of interest. i These two metrics, ΔTavg and ΔTgrad, are calculated for the conditions explored so far: 1.5C constant current discharge, with 25 ◦C ambient and cooling plate temperature, under conductive cooling of top & base and side, and under convective cooling at top & base and side, characterized by 30 Wm−2K−1, with all other surfaces insulated. The results for the 2170 and 4680 cells are plotted in Fig. 9 and quantitatively compared in Table 3. When the 4680 cell is cooled from the top and base by con vection or conduction, both ΔTavg and ΔTgrad are significantly lower than those for the 2170 cell under the same conditions, as listed in Table 3. The best thermal performance of the 4680 cell (ΔTavg = 5.62◦C, ΔTgrad = 7.61◦C) is similar to the best thermal performance of the 2170 cell (ΔTavg = 4.81◦C, ΔTgrad = 8.23◦C). The former is achieved under top and base conduction cooling, while the latter under side conduction In order to compare the different cells, tab configurations, and thermal management options, we have used the ‘average functions’ defined in our previous work [13]. As described by Li et al. [13], ΔTavg 11 Applied Energy 349 (2023) 121548 S. Li et al. Fig. 9. Predicted thermal performance of the 2170 cell and the 4680 cell under top/base cooling and side cooling approaches. The 2170 cell side conduction cooling and the 4680 cell top/base conduction cooling performance are very similar, indicating the optimum cooling approaches for these cells. dimension and cooling approach, remain unchanged. Once the cell is selected, the cooling system should be designed according to the con dition that minimizes the two metrics. 5. Discussion In this manner, the combination of chosen cell design and thermal management will maximize the cell performance and thus extend the battery system lifetime. Table 3 This result demonstrates there may not need to be a trade-off between achieving high energy density and good thermal perfor mance, as long as cylindrical cells can be built with a tabless design. Under side cooling, the 2170 cell performs close to the 4680 cell with respect to both ΔTavg and ΔTgrad. For the 4680 cell, the best cooling approach is conductive top/base cooling; for the 2170 cell, it is side conductive cooling, as it does not generate a high internal thermal gradient. The surface-to-volume ratio for the 2170 cell is roughly double that of the 4680 cell, 2.19 × 102 m−1 and 1.12 × 102 m−1 respectively. The high surface-to-volume ratio of the 2170 cell maximizes the heat extraction efficiency and maintains a low volume-averaged tempera ture. The relatively smaller radius of the 2170 cell enables it to avoid high thermal gradients, which do occur in the 4680 cell, as listed in in Table 3: for side convection the 4680 cell has a thermal gradient higher by roughly a factor of two, while for side conduction by roughly a factor of three. The study unveils that the ability to manufacture with a continuous (or tabless) tab configuration is essential to creating viable large form factor cylindrical cells. Importantly, this is one example of significant advances in cell design from a thermal management point of view, creating a net positive impact on cell performance, despite increasing the mass of the cell. This demonstrates there does not need to be a trade- off between cell size and thermal performance, as long as cylindrical cells are designed tabless. 6. Conclusions A distributed 3D coupled electro-thermal equivalent circuit network (ECN) model of cylindrical lithium-ion batteries is used to study the effect of cell design and cooling approach on performance. Multiple tab configurations and thermal management approaches are considered for 2170 and 4680 cells. The distributed ECN model includes the non-active components, with particular attention to correctly describing the ther mal paths within the cell. The internal thermal connection between the jellyroll and the metal can is found to be essential in determining the performance of the cell, despite it being ignored in most published battery modelling studies. i The model was validated against base cooling cell cooling coefficient (CCC) experimental tests under various SoC, current and temperature conditions on a 2170 single tab cell, the LG M50T. Once validated, the model was used to explore the capabilities of a tabless large form factor 4680 cylindrical cell, for example as the one developed by Tesla. The simulation results show that the tabless design significantly improves both the electrical and thermal performance of a cylindrical cell. Using base cooling, the normalized cell cooling coefficient for the 4680 tabless cell is almost twice that of the non-tabless 2170 single-tab and of the 4680 all-tab cells. This improvement is found to be caused by the shortened electrical path and the enhanced internal metal-metal thermal connection between the jellyroll and the metal can, at both the negative and positive terminals. The results also show that a 4680, or other large format cylindrical cell, would not be viable without a tabless design, as they would either reach the voltage cut-off too early due to high re sistances, or overheat. i Fig. 9. Predicted thermal performance of the 2170 cell and the 4680 cell under top/base cooling and side cooling approaches. The 2170 cell side conduction cooling and the 4680 cell top/base conduction cooling performance are very similar, indicating the optimum cooling approaches for these cells. Table 3 cooling. This result demonstrates there may not need to be a trade-off between achieving high energy density and good thermal perfor mance, as long as cylindrical cells can be built with a tabless design. Under side cooling, the 2170 cell performs close to the 4680 cell with respect to both ΔTavg and ΔTgrad. For the 4680 cell, the best cooling approach is conductive top/base cooling; for the 2170 cell, it is side conductive cooling, as it does not generate a high internal thermal gradient. The surface-to-volume ratio for the 2170 cell is roughly double that of the 4680 cell, 2.19 × 102 m−1 and 1.12 × 102 m−1 respectively. The high surface-to-volume ratio of the 2170 cell maximizes the heat extraction efficiency and maintains a low volume-averaged tempera ture. The relatively smaller radius of the 2170 cell enables it to avoid high thermal gradients, which do occur in the 4680 cell, as listed in in Table 3: for side convection the 4680 cell has a thermal gradient higher by roughly a factor of two, while for side conduction by roughly a factor of three. Table 3 Thermal metric values (ΔTavg \| ΔTgrad, unit: ◦C) for the 2170 and the 4680 cells under top/base and side cooling, as plotted in Fig. 9. Top/base conv. Top/base cond. Side conv. Side cond. 2170 single- tab 86.84 \| 20.92 33.33 \| 35.66 39.07 \| 5.66 4.81\| 8.23 4680 tabless 67.77 \| 2.64 5.62\| 7.61 49.74 \| 12.44 13.23\| 23.02 A procedure for finding the best thermal management for cylindrical cells is proposed by defining two thermal metrics. For the 4680 tabless cell compared to the 2170 cell, the thermal metrics of average temper ature and internal thermal gradient are found to be no worse, while the 4680 tabless tab cell has 5.4 times the energy and 6.9 times the power. The results show that the best cooling strategy for the single-tab 2170 is side cooling, while for the 4680 tabless cell it is base cooling. The op timum cooling strategy is strongly affected by both the surface-to- volume ratio and the internal thermal connection of the cell. The surface-to-volume ratio dominates for the single-tab 2170, leading to side cooling, while the enhanced thermal connection between the jel lyroll and metal can dominates for the 4680 tabless cell leading to base cooling. cooling. References org/10.1149/2.1241810jes. [8] Hua X, Heckel C, Modrow N, Zhang C, Hales A, Holloway J, et al. 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Monica Marinescu: Conceptualization, Formal analysis, Funding acquisition, Methodology, Supervision, As an immediate implication to battery engineering, the two metrics ΔTavg and ΔTgrad should lead the cell selection process, itself an important process for both the stationery energy storage system (ESS) and the electric vehicle (EV) industries. We recommend that the best cell is selected as the one that has lowest values for the two characteristics, as calculated from an appropriate model. If practical, a tabless design is shown to lead to better performance when other parameters, such as cell 12 S. Li et al. Applied Energy 349 (2023) 121548 Writing – original draft, Writing – review & editing. [17] Tsuruta K, Dermer ME, Dhiman R. Cell with a tabless electrode, 16/673464. http s://patents.google.com/patent/US20200144676A1/en/; 2020. [18] Tranter TG, Timms R, Shearing PR, Brett DJL. Communication—prediction of thermal issues for larger format 4680 cylindrical cells and their mitigation with enhanced current collection. J. Electrochem. Soc. 2020;167:160544. https://doi. org/10.1149/1945-7111/abd44f. Data will be made available on request. [21] Erhard SV, Osswald PJ, Wilhelm J, Rheinfeld A, Kosch S, Jossen A. Simulation and measurement of local potentials of modified commercial cylindrical cells. J. Electrochem. Soc. 2015;162:A2707–19. https://doi.org/10.1149/2.0431514jes. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. [19] Worwood D, Kellner Q, Wojtala M, Widanage WD, McGlen R, Greenwood D, et al. A new approach to the internal thermal management of cylindrical battery cells for automotive applications. J. Power Sources 2017;346:151–66. https://doi.org/ 10.1016/j.jpowsour.2017.02.023. [20] Sturm J, Rheinfeld A, Zilberman I, Spingler FB, Kosch S, Frie F, et al. Modeling and simulation of inhomogeneities in a 18650 nickel-rich , silicon- graphite lithium-ion cell during fast charging. J. Power Sources 2019;412:204–23. https://doi.org/ 10.1016/j.jpowsour.2018.11.043. Acknowledgements [22] Hales A, Marzook MW, Bravo Diaz L, Patel Y, Offer G. The surface cell cooling coefficient: a standard to define heat rejection from lithium ion battery pouch cells. J. Electrochem. Soc. 2020;167:020524. https://doi.org/10.1149/1945-7111/ ab6985. This work was generously supported by the EPSRC Faraday Institu tion Multi-Scale Modelling project (EP/S003053/1, grant number FIRG003), the Innovate UK WIZer project (grant number 104427) and the EPSRC CASE (grant number EP/R513052/1) award by Williams Advanced Engineering. We thank Dr. Tazdin Amietszjew for providing the picture in Fig. 1(d). [23] Lander L, Kallitsis E, Hales A, Edge JS, Korre A, Offer G. Cost and carbon footprint reduction of electric vehicle lithium-ion batteries through efficient thermal management. Appl. Energy 2021;289:116737. https://doi.org/10.1016/j. apenergy.2021.116737. [24] Marzook MW, Hales A, Patel Y, Offer G, Marinescu M. Thermal evaluation of lithium-ion batteries : defining the cylindrical cell cooling coefficient. J Energy Storage 2022;54:105217. https://doi.org/10.1016/j.est.2022.105217. Data availability Data will be made available on request. References 3D electro-thermal model approach for the prediction of internal state values in large-format lithium ion cells and its validation. J. Electrochem. Soc. 2014;161:A1943–52. https://doi.org/ 10.1149/2.1201412jes. [14] Lee KJ, Smith K, Pesaran A, Kim GH. Three dimensional thermal-, electrical-, and electrochemical-coupled model for cylindrical wound large format lithium-ion batteries. J. Power Sources 2013;241:20–32. https://doi.org/10.1016/j. jpowsour.2013.03.007. l j [42] Inc Tesla. 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W4293370778.txt	https://www.revista.ccba.uady.mx/ojs/index.php/TSA/article/download/3457/1626	en		DISEÑO Y EVALUACIÓN DE MEDIOS LÍQUIDOS PARA LA PRODUCCIÓN DE BLASTOSPORAS DE Beauveria bassiana	Tropical and subtropical agroecosystems	2,021	cc-by	5,253	Tropical and Subtropical Agroecosystems 24 (2021): #80 Elías-Santos et al., 2021 DISEÑO Y EVALUACIÓN DE MEDIOS LÍQUIDOS PARA LA PRODUCCIÓN DE BLASTOSPORAS DE Beauveria bassiana † [DESIGN AND EVALUATION OF LIQUID MEDIA FOR THE PRODUCTION OF BLASTOSPORES OF Beauveria bassiana] Myriam Elías-Santos1, Jairo H. Alfaro-Álvarez1, Isela Quintero-Zapata1, Hugo A. Luna-Olvera1, Benito Pereyra-Alférez1, Luis J. Galán-Wong1, María Guadalupe Maldonado-Blanco1, Claudio Guajardo-Barbosa1, José Lorenzo Meza-García2 and Fatima Lizeth Gandarilla-Pacheco1* 1 Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, Instituto de Biotecnología. Pedro de Alba s/n, Cd. Universitaria, San Nicolás de los Garza, N.L. México, C.P. 66450. Email: myriam.eliassn@uanl.edu.mx, jairo.alvarez@hotmail.it, isela.quinterozp@uanl.edu.mx, hugo.lunaol@uanl.edu.mx, benito.pereyraal@uanl.edu.mx, luis.galanwn@uanl.edu.mx, maria.maldonadobl@uanl.edu.mx, cguajardob@hotmail.com, fatimagandarilla84@gmail.com 2 Universidad Autónoma de Occidente, Unidad Regional Guasave. Av. Universidad s/n, Fraccionamiento Villa Universidad, Guasave, Sinaloa, México, C.P. 81000. Email. lorenzo_meza@prodigy.net.mx Corresponding author SUMMARY Background. The nitrogen source in a liquid culture medium for the propagation of entomopathogenic fungi is considered an essential element in fermentation, however, it is one of the most expensive, which makes the search for alternative sources of this nutrient important. Objective. This research compared the production of blastospores obtained by designing different culture media for the GHA strain of Beauveria bassiana based on the nitrogen source used. Methodology. Six media were designed in liquid form, made with various nitrogen sources (sesame flour, amaranth flour, canary seed flour, linseed flour, peanut pericarp flour, oatmeal) with glucose corn as a carbon source for all of them. Additionally, the yield of blastospores obtained at different incubation times, the pH and the viability of the post-fermentation propagules were evaluated. Results. The M8 medium made with peptone and yeast extract as nitrogen sources was the one that obtained the highest concentration with 1.28 × 109 blastospores / mL at 168 hours of incubation. Regarding the fermentation parameters evaluated, the pH ranged between 5.1- 5.5 and the determination of this parameter did not present significant difference with respect to the culture medium used or the day on which it was determined (p ≥ 0.05) while post-fermentation viability was determined. In two solid media, and according to the results in potato dextrose agar the viability ranged between 31-90% on average while in water agar it was 25-90%, the determination of this parameter did not present significant difference with respect to the medium of culture for production of blastospores or to the solid medium in which it was determined (p ≥ 0.05). Implications. The alternate nitrogen sources used did not significantly increase the production of blastospores compared to conventional sources such as peptone and yeast extract, which in a synergistic effect did manage to enhance the production of blastospores. Conclusion. The use of yeast extract and casein peptone in conjunction can increase the yield of blastsopores in a liquid culture medium. Keywords: Beauveria bassiana; blastospores; nitrogen source; glucose. RESUMEN Antecedentes. La fuente de nitrógeno en un medio de cultivo líquido para la propagación de hongos entomopatógenos se considera un elemento esencial en la fermentación, sin embargo, es uno de los más costosos, lo cual hace importante la búsqueda de fuentes alternativas de este nutriente. Objetivo. Esta investigacióncomparó la producción de blastosporas obtenida mediante el diseño de diferentes medios de cultivo para la cepa GHA de Beauveria bassiana con base en la fuente de nitrógeno utilizada. Metodología. Se diseñaron seis medios en forma líquida, elaborados con diversas fuentes de nitrógeno (harina de ajonjolí, harina de amaranto, harina de alpiste, harina de linaza, harina de pericarpio de cacahuate, harina de avena) y con glucosa de maíz como fuente de carbono para todos ellos. Adicionalmente se evaluó el rendimiento de blastosporas obtenidas a diferentes tiempos de † Submitted August 17, 2020 – Accepted October 18, 2020. This work is licensed under a CC-BY 4.0 International License. ISSN: 1870-0462. 1 Tropical and Subtropical Agroecosystems 24 (2021): #80 Elías-Santos et al., 2021 incubación, el pH y la viabilidad de los propágulos post fermentación. Resultados. El medio M8 elaborado con peptona y extracto de levadura como fuentes de nitrógeno fue el que obtuvo la mayor concentración con 1.28 × 109 blastosporas/mL a las 168 horas de incubación. Respecto a los parámetros de fermentación evaluados el pH osciló entre 5.1- 5.5 y la determinación de este parámetro no presentó diferencia significativa respecto al medio de cultivo utilizado ni al día en que fue determinado (p ≥ 0.05) mientras que la viabilidad post fermentación se determinó en dos medios sólidos, y de acuerdo a los resultados en agar papa dextrosa la viabilidad oscilo entre 31-90 % en promedio mientras que en agar agua fue de 25-90%, la determinación de este parámetro no presentó diferencia significativa respecto al medio de cultivo para la producción de blastosporas ni al medio sólido en que fue determinado (p ≥ 0.05). Implicaciones. Las fuentes alternas de nitrógeno utilizadas no aumentaron en forma significativa la producción de blastosporas en comparación con las fuentes convencionales como la peptona y elextracto de levadura, las cuales en un efecto sinérgico si lograron potenciar la producción de blastosporas. Conclusión. El uso de extracto de levadura y peptona de caseína en conjunción, pueden aumentar el rendimiento de blastosporas en un medio de cultivo líquido. Palabras clave: Beauveria bassiana; blastosporas; fuente de nitrógeno; glucosa. microesclerocios (Mascarin et al., 2015; HuarteBonnet et al., 2019). B. bassiana actualmente se comercializa como ingrediente activo en diferentes micoinsecticidas, ocupando un 33.9% del mercado aproximadamente, y son los conidios aéreos el ingrediente activo principal de estos productos (de Faria & Wraight, 2007). La producción de uno u otro propágulo va a estar sujeta a diferentes factores que tienen que ver con los métodos de producción, medios de propagación, incluso tomando en cuenta el tipo de cultivo donde se aplicarán y la plaga de interés (Gandarilla-Pacheco et al., 2018), sin embargo, las blastosporas se consideran una opción viable debido a que presentan ciertas ventajas frente a los conidios como propágulo de elección. Por ejemplo, la producción de blastosporas puede obtenerse en cortos tiempos de fermentación de hasta 3 días, facilidad en la recuperación y una automatización de los procesos (Jackson ,1997). INTRODUCCIÓN Recientemente se ha considerado a la agricultura como uno de los sectores económicos más importantes a nivel global. Estudios recientes estiman que la población mundial podría alcanzar hasta 10 billones de personas para el año 2050. Como una solución a la demanda de alimentos se han utilizado fertilizantes y plaguicidas para incrementar la producción de cultivos con fines alimentarios (Bautista et al., 2018), así como la protección de estos cultivos del ataque de insectos plaga, sin embargo, el uso indiscriminado de estos productos ha ocasionado problemas de contaminación ambiental y desarrollo de resistencia en insectos plaga, por mencionar algunos de sus efectos nocivos. En este sentido, el uso de plaguicidas de origen biológico se ostentan como una opción para el control de plagas, debido a que estos productos presentan baja residualidad y generalmente muestran especificidad hacia su blanco, reduciendo así el desarrollo de resistencia (Arthurs & Dara, 2019). Esta constante de la degradación ambiental causada por las prácticas agrícolas convencionales ha permitido que el empleo de hongos entomopatógenos como control biológico se generalice cada vez más (Baron et al., 2019). Los hongos entomopatógenos son microorganismos con la capacidad de infectar y matar artrópodos. Se utilizan principalmente como bioplaguicidas en la agricultura ecológica como una alternativa a los insecticidas químicos (Litwin et al., 2020). De acuerdo a Mishra et al (2015) los hongos ocupan el segundo lugar en el mercado mundial dentro de los plaguicidas microbianos, mientras que en América Latina se utilizan en mayor medida los micoplaguicidas (Mascarin et al., 2018). Beauveria bassiana es una especie ampliamente distribuida, este hongo se considera ubicuo capaz de explotar una variedad de entornos, incluidos el suelo, las plantas y los insectos; puede vivir como un saprófito en el suelo, o como un entomopatógeno que afecta a una amplia gama de artrópodos (Zimmerman, 2007; Boomsma et al., 2014). B. bassiana presenta diferentes tipos de propágulos, específicamente, conidios, blastosporas y En el desarrollo de un proceso de producción para la obtención de un micoplaguicida se deben cumplir diferentes etapas, primeramente la selección de la cepa en base a su virulencia, su capacidad de resistir condiciones ambientales adversas relacionadas con la resistencia a la luz UV, tolerancia a la temperatura, en segundo la selección de un medio de cultivo optimo que pueda ser de bajo costo o el uso de substratos alternativos, en tercero y relacionado con el medio de cultivo es la elección del método de producción , así como los procesos de separación, los parámetros a controlar durante el proceso,control de calidad y costos (Pourseyed et al., 2010; Bautista et al., 2018). Los hongos entomopatógenos pueden obtenerse mediante diversas técnicas de propagación como la fermentación sólida, cultivo líquido o sumergido y cultivo bifásico. Todas las técnicas varían entre ellas y tienen ventajas o desventajas según el propágulo y la cantidad que desea obtener, por ejemplo, el cultivo liquido puede facilitar el proceso de escalamiento además de garantizar la producción de propágulos bajo condiciones controladas (Jackson et al., 2003). En la fermentación en sustrato solido la cual puede resultar atractiva a primera vista por los bajos costos 2 Tropical and Subtropical Agroecosystems 24 (2021): #80 Elías-Santos et al., 2021 de los sustratos, que en ocasiones pueden ser hasta residuos de procesos agroindustriales, el proceso de producción requiere semanas y existe el riesgo constante de contaminación (Mascarin et al., 2015). Incluso y con todas sus bondades la fermentación liquida no está exenta de presentar algunas dificultades en su implementación, la más común es el costo de los medios de cultivo utilizados a comparación de los empleados en la fermentación sólida, por lo tanto, es importante la búsqueda de sustratos de menor costo en la optimización de este proceso. La fuente de carbono, nitrógeno, metales, vitaminas y la relación entre estos elementos, pueden influir en el crecimiento, la formación de propágulos y la eficacia (Hegedus et al., 1990). Desde hace décadas se conoce que los elementos más abundantes en las células fúngicas son el carbono, oxígeno y nitrógeno, siendo este último uno de los más críticos en la fermentación debido a su alto costo (Mascarin et al., 2015). componentes para cada uno de los medios se resume en la Tabla 1. Preparación de las harinas utilizadas como fuente de nitrógeno Las harinas se obtuvieron de manera artesanal a partir de cinco tipos de granos (ajonjolí, alpiste, avena, linaza y pericarpio de cacahuate). Los granos se procesaron en una licuadora hasta obtener un polvo fino, el cual se tamizó en una malla de 212 µm hasta obtener la consistencia de una harina típica. Las harinas obtenidas se almacenaron en recipientes plásticos a temperatura ambiente hasta su uso. Obtención del inoculo La cepa GHA se cultivó durante 14 días en agar papa dextrosa a 25 ± 2°C. Posteriormente con ayuda de un asa Drigalsky se raspó la superficie de las cajas para desprender los conidios, para facilitar este proceso se agregaron 20 mL agua bidestilada estéril, finalmente este volumen se colectó en un frasco donde se diluyó y homogenizópara así obtener un volumen final de 100 mL. Finalmente se ajustó la suspensión a 5 × 105 conidios/mL mediante un recuento en cámara de Neubauer para la posterior inoculación del cultivo líquido-sumergido. Esta situación hace imperativa la búsqueda de sustratos de bajo costo para optimizar procesos y obtener propágulos de hongos que sean eficaces contra insectos plaga y en cantidades suficientes. El objetivo de este estudio fue diseñar diferentes medios de cultivo líquido para obtener blastosporas de Beauveria bassiana mediante el uso de fuentes alternativas de nitrógeno. Condiciones de fermentación MATERIALES Y MÉTODOS Para el cultivo se utilizaron matraces con bafles de 250 mL de capacidad, se les colocó 45 mL de cada uno de los medios y 5 mL de la suspensión del hongo para obtener un volumen total de 50 mL. Los matraces se agitaron a 250 rpm y se inocularon a 28° C. Los recuentos de blastosporas se realizaron a las 72, 120 y 168 horas de incubación. Microorganismos Beauveria bassiana GHA (ARSEF 6444), el ingrediente activo del micoinsecticida comercializado bajo el nombre de Mycotrol®, fue la cepa utilizada en esta investigación. Este microorganismo fue donado a la colección del laboratorio L6 del Instituto de Biotecnología con sede en la Facultad de Ciencias Biológicas de la Universidad Autónoma de Nuevo León (FCB-UANL). Para su conservación esta cepa se encontraba en una solución de glicerol al 10 % (v/v) a -80 ° C. Determinación del pH Se determinó para cada uno de los medios evaluados al finalizar el proceso de fermentación; para lo cual se utilizó un potenciómetro digital (Beckman OHMS 50 pH meter) escala 1-14, previamente una calibración del mismo; se tomó 1 mL de cada una de los tratamientos y se depositó en tubos Falcon para la medición del potencial de hidrógeno. Medios de cultivo Para la obtención de las blastosporas se diseñaron seis medios de cultivo los cuales contenían una sola fuente de carbono (glucosa de maíz al 20 % (p/v) y diversas harinas procedentes de granos de cereales y residuos como fuentes de nitrógeno (ajonjolí, alpiste, amaranto, avena, linaza y pericarpio de cacahuate). Adicionalmente se agregaron cuatro medios como controles positivos suplementados con sales inorgánicas (CaCO3, NaCl, KH2PO4, MgSO4, CaCl2, FeSO4) y soluciones metálicas (ZnSO4, MnSO4, CoCl2) y uno más como control negativo el cual solo contenía la fuente de carbono. La proporción de los Viabilidad de blastosporas La viabilidad post fermentación se realizó en placas Petri previamente cuadriculadas que contenían agar papa dextrosa y agar agua, al solidificar se colocó una gota de las muestras diluidas a 10-1 y 10-2 en cada uno de los medios. A las diez horas se cortó con un bisturí cada cuadro y se colocó una gota de azul de lactofenol, se contaron 100 blastosporas en un microscopio óptico con el objetivo de 40 X para 3 Tropical and Subtropical Agroecosystems 24 (2021): #80 determinar la presencia o ausencia del germinativo. Elías-Santos et al., 2021 tubo incubación en los cuales se reportó una diferencia significativa (p=0.001) en la producción de blastosporas de acuerdo al medio de propagación. Esta tendencia se mantuvo a las 120 y 168 horas de incubación donde se reporta una diferencia altamente significativa en la obtención de blastosporas entre cada medio (p=0.00001) y se observó que el medio M8 es el que alcanzó el mayor número de blastosporas (Tabla 2). Cinética de crecimiento a nivel matraz Este procedimiento se realizó exclusivamente con el medio seleccionado con la mayor producción de blastosporas. Se tomaron muestras cada 48 h, durante 14 días. Estas muestras fueron separadas y se les realizó un conteo de blastosporas para construir una curva de crecimiento. En la medición del pH dicho parámetro se mantuvo en un rango de 5.1 a 5.6 y de acuerdo a los resultados estadísticos no presentó diferencia significativa respecto al medio de cultivo (p= 0.672) ni al día en que fue determinado (p=0.400) (Figura 1). Análisis estadístico El diseño de los experimentos fue completamente al azar, con cuatro repeticiones por tratamiento. Los resultados obtenidos se reportan como promedios incluyendo el valor del error estándar. Los resultados se convirtieron a Log10 para procesarlos en el programa estadístico IBM SPSS® v.19 y mediante la prueba de Kolmogorov-Smirnov para verificar la normalidad de los datos. Posteriormente se realizó un análisis de varianza (ANOVA) y una prueba de Tukey (p≤0.05). Los experimentos fueron repetidos en al menos dos ocasiones. El porcentaje de germinación en agar papa dextrosa osciló entre 31-90 %, mientras que en agar agua se registró de 25- 90 % y de acuerdo con los resultados estadísticos la determinación de este parámetro no presentó diferencia significativa respecto al medio de cultivo para la producción de blastosporas (p= 0.368) ni al medio sólido en que fue determinado (p=0.438) (Figura 2). La curva de crecimiento de la cepa GHA se determinó en el medio M8 elaborado con peptona de caseina y extracto de levadura por ser el que obtuvo mayor producción de blastosporas. Los resultados obtenidos muestran que a las 48 horas se obtuvo el nivel más RESULTADOS Y DISCUSIÓN La cantidad de blastosporas en cada uno de los medios líquido aumentó a partir de las 72 horas de Tabla 1. Composición de los medios de cultivo para la propagación de Beauveria bassiana. Control Tratamientos Control (-) (+) Sustrato M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 Glucosa de maíz (mL) 200 200 200 200 200 200 200 200 200 200 Harina de ajonjolí (g) 20 Harina de alpiste (g) 20 Harina de amaranto (g) Harina de avena (g) Harina de pericarpio de cacahuate (g) Harina de linaza (g) Extracto de levadura (g) CaCO3 (g) NaCl (g) Peptona (g) KH2PO4 (g) MgSO4 (g) CaCl2 (g) FeSO4 (g) ZnSO4 (mL) MnSO4 (mL) CoCl2 (mL) *Cada formulado corresponde a la cantidad de un litro. M11 120 20 20 20 20 5 1 5 20 4 5 1 5 5 5 4 0.6 0.8 0.1 4 0.6 0.8 0.1 60 60 60 Tropical and Subtropical Agroecosystems 24 (2021): #80 Elías-Santos et al., 2021 Tabla 2. Blastosporas de Beauveria bassiana en cultivo líquido a distintos tiempos de incubación en los medios evaluados. Medio Blastosporas/mL M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 Media ± EE EE: Error estándar. 72 horas 2.15 × 107(a) 6.05 × 107(ab) 3.05 × 107 (ab) 9.20 × 107(ab) 8.41 × 107( ab) 2.20 × 107( a ) 6.70 × 107(ab) 2.10 × 108(b) 1.98 × 108( b) 1.42 × 108(ab) 1.04 × 108(ab) 7.00 × 107 ± 0.10 120 horas 3.95 × 107 (a) 1.08 × 108 (bcd) 9.78 × 107 (abc) 2.10 × 108 (cde) 2.08 × 108 (cde) 4.35 × 107 (ab) 1.78 × 108 (cde) 9.15 × 108 (g) 7.25 × 108 (fg) 2.67 × 108 (de) 8.51 × 108 (ef) 1.82 × 108 ± 0.13 168 horas 3.64 × 107 (a) 1.22 × 108 (bc) 1.13 × 108 (abc) 2.29 × 108 (cd) 1.78 × 108(cd) 5.10 × 108 (ab) 1.65 × 108 (cd) 1.28 × 109 (f) 9.80 × 108 (ef) 2.70 × 108 (cd) 3.91 × 108 (de) 2.00 × 108 ± 0.14 Figura 1. pH promedio de los medios para la producción de blastosporas de Beauveria bassiana (GHA) a diferentes tiempos de incubación en cultivo sumergido a nivel matraz bajo condiciones de laboratorio (28 °C; 250 RPM). bajo con 6.90 × 107 blastosporas/mL, mientras que a las 96 horas de incubación se obtuvieron 9.20 × 108 blastosporas/mL, y a las 144 horas la produccion aumentó significativamente hasta 2.43 × 109 blastosporas/mL, y siguio aumentando exponencialmente hasta alcanzar 3 × 109 blastosporas/mL (Figura 3). El interés de estudiar las condiciones para producir masivamente propágulos de B. bassiana datan de hace varias décadas y con los avances tecnológicos consecuentes estos métodos se han ido perfeccionando. Distintos autores han documentado métodos utilizados en la optimización para la obtención de blastosporas de B. bassiana en cultivo liquido haciendo énfasis en las variaciones en el medio de cultivo y condiciones de la fermentación 5 Tropical and Subtropical Agroecosystems 24 (2021): #80 Elías-Santos et al., 2021 (Rombach et al., 1989; Chong- Rodríguez et al., 2011; Sandoval-Coronado et al., 2010; GandarillaPacheco et al., 2013; Mascarin et al., 2015). En este trabajo se diseñaron once medios para la propagación a nivel matraz de blastosporas de la cepa GHA de B. bassiana, con diferentes fuentes de nitrógeno y se obtuvieron valores promedio de 107- 109 blastosporas/mL. El nitrógeno es un elemento clave en los procesos fermentativos (Mascarin et al., 2018) es por eso que en los últimos años la tendencia es la búsqueda de nuevas fuentes, orgánicas o inorgánicas, de nitrógeno que tengan como característica disponibilidad y bajo costo en el mercado con la finalidad de optimizar recursos en la elaboración de medios de cultivo para fermentación. Estudios anteriores se han planteado esta problemática y han obtenido resultados similares a los de este trabajo, Chong- Rodríguez (2011) determinó que un medio a base de glucosa y peptona como fuente de nitrógeno alcanzó niveles de 1.60× 109 blastosporas/mL para la cepa GHA, estos resultados concuerdan con un estudio reciente con la misma cepa donde utilizaron la harina de semilla de algodón como fuente de nitrógeno alcanzando una producción de 1.46 × 109 blastosporas/mL (Mascarin et al., 2018), mientras que en el presente trabajo la mayor producción de blastosporas se alcanzó con el medio que contenía peptona y extracto de levadura, mientras que los medios donde se utilizaron harinas solo alcanzaron niveles de 108 blastosporas/mL, lo cual coincide con lo reportado en un trabajo anterior donde determinaron la producción de blastosporas en un medio de casaminoácidos y glucosa para la cepa GHA alcanzando una producción de 2.20 × 108 blastosporas/mL a las 72 horas (Gandarilla-Pacheco et al., 2013). Es interesante resaltar las variaciones existentes en estos trabajos pese a que la cepa en estudio es la misma, esta variabilidad puede deberse a distintos factores que pueden incidir directamente sobre la producción de blastosporas como la cantidad de inoculo inicial, el tipo y velocidad de agitación, temperatura y el mismo medio de cultivo. Por otro lado, Kleespies y Zimmermann (1992) mencionan que los hongos entomopatógenos son poseedores de una variabilidad genética amplia y pueden presentar diferencias cuando se propagan en cultivo líquido, por lo que se considera importante lograr la optimización de los parámetros de producción en medios de cultivo líquido para cada cepa en específico. Figura 2. Porcentaje de germinación en agar papa dextrosa (PDA) y agar agua (AA) como sustratos de las blastosporas de Beauveria bassiana (GHA) obtenidas post fermentación en cultivo sumergido a nivel matraz bajo condiciones de laboratorio (25 ± 2 °C; 12:12 h luz: oscuridad). 6 Tropical and Subtropical Agroecosystems 24 (2021): #80 Elías-Santos et al., 2021 Figura 3. Cinética de crecimiento de la cepa GHA de B. bassiana en el medio de cultivo M8 a nivel matraz bajo condiciones de laboratorio (28° C; 250 RPM). Generalmente la producción de blastosporas a nivel laboratorio se lleva a cabo en equipos de agitación orbital como el utilizado en este estudio, sin embargo, Villalba et al. (2010) mencionan que la agitación mecánica mostro una concentración de blastosporas de hasta veinte veces más que en el sistema de agitación orbital para una cepa de B. bassiana por lo cual se puede considerar como un factor, aunque en el presente estudio no se realizó la producción en diferentes equipos. de levaduras seleccionadas. Este producto contiene vitaminas del complejo B, aminoácidos y algunos factores de crecimiento, mientras que las peptonas son una mezcla hidrosoluble de polipéptidos, péptidos, aminoácidos y otras sustancias que quedan después de la digestión del material proteico. La calidad de las peptonas viene determinada por la calidad de las materias primas seleccionadas, sus condiciones de almacenamiento y los parámetros de la digestión. En este trabajo se utilizó la peptona de caseína que es un digerido pancreático de caseína que está compuesto por una mezcla de aminoácidos y péptidos de bajo peso molecular, sin embargo, Griffin (1996) menciona que este tipo de peptona no presenta un contenido aceptable de vitaminas lo cual puede incidir en la esporulación. En este trabajo esta carencia parece haber sido suplementada con la adición del extracto de levadura. Mascarin et al (2018) mencionan que la disposición de nitrógeno como aminoácidos en su forma libre influye directamente en la producción de blastosporas y la resistencia a la desecación, aunque el incremento de la concentración de glucosa y nitrógeno hasta 120 g/L y 30 g/L, respectivamente, no mejora o eleva la producción de blastosporas, si aumentó la tolerancia a la desecación lo cual se considera un parámetro crítico para la formulación. En este trabajo las fuentes de nitrógeno que promovieron la mayor producción de blastosporas fueron el extracto de levadura y la peptona, lo que es similar a lo reportado por Sandoval-Coronado et al (2010) quienes reportan para la cepa GHA una producción de 5.1×109 blastosporas/mL en un medio de cultivo con 40 g/L de glucosa y 6 g/L de peptona de colágena a las 120 h. El extracto de levadura es un autolisado de células Respecto a las harinas utilizadas, estas no mostraron diferencias significativas en la producción de blastosporas. Estas harinas procedentes de granos de acuerdo con tablas nutrimentales contienen en promedio desde 13 a 18 gramos de proteína por cada 100 gramos (FUNIBER, 2020), sin embargo, BaduiDergal (1993) menciona que una proteína puede ser altamente nutritiva y carecer de funcionalidad en un proceso. Otro criterio que se debe considerar cuando 7 Tropical and Subtropical Agroecosystems 24 (2021): #80 Elías-Santos et al., 2021 se utilizan cereales es su composición química, la cual puede variar dependiendo de los factores ambientales. en las instalaciones de la Universidad Autónoma de Nuevo León. Disponibilidad de datos. Los datos están disponibles con el autor correspondiente (fatimagandarilla84@gmail.com) bajo previa solicitud debidamente justificada. Otra de las limitantes en el uso de granos de cereales como sustratos para la producción de hongos radica en el hecho de que existen fracciones resistentes a la digestión enzimática. Este fenómeno se ha documentado para el almidón el cual presenta en su estructura una fracción resistente que se mantiene integra durante el paso por el tracto gastrointestinal humano (Villaroel et al., 2018). En el caso de la presente investigación las semillas utilizadas para la elaboración de las harinas empleadas en los medios de cultivo evaluados no se sometieron a ningún pretratamiento de digestión enzimática lo cual pudo haber influido en la disponibilidad de las mismas y evitó que pudieran ser metabolizadas adecuadamente por B. bassiana. Esta hipótesis se apoya en el hecho de que la mayor producción de blastosporas se observó en el medio que contenía extracto de levadura y peptona de caseína debido a que ambos compuestos son un autolisado celular y un digerido pancreático, respectivamente, lo cual significa que al estar hidrolizados se aumenta la posibilidad de que sea metabolizado por el organismo en cuestión sin que esto signifique un gasto de energía adicional debido al pretratamiento enzimático. REFERENCIAS Arthurs, S. and Dara, S.K. 2019. Microbial biopesticides for invertebrate pests and their markets in the United States. 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CONCLUSIONES Los resultados de este trabajo muestran la factibilidad de utilizar fuentes alternativas de nitrógeno para inducir la producción de blastosporas de B. bassiana mediante cultivo sumergido, aunque las harinas probadas se encontraron por debajo de la producción alcanzada con la peptona y la levadura de cerveza, estas se mantuvieron en el rango de 108 blastosporas/mL y estos rendimientos podrían ser mejorados en futuros ensayos modificando las proporciones C:N e incluyendo pretratamientos por digestión enzimática para aumentar la biodisponibilidad de los nutrientes. Boomsma, J.J., Jensen, A.B., Meyling, N.V., and Eilenberg, J. 2014.Evolutionary interaction networks of insect pathogenic fungi. Annual Review of Entomology, 59, pp. 467-485. https://doi.org/10.1146/annurev-ento011613-162054. Chong-Rodríguez, M.J, Maldonado-Blanco, M.G., Hernández-Escareño, J.J., Galán-Wong, L.J. and Sandoval-Coronado, C.F. 2011. 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Biocontrol Science and Technology, 2, pp. 127-135. https://doi.org/10.1080/09583159209355226. Litwin, A., Nowak, M. and Rózalska, S. 2020. Entomopathogenic fungi: unconventional applications. Reviews in Environmental Science and Biotechnology, 19, pp. 23-42. https://doi.org/10.1007/s11157-020-09525-1. Mascarin, G.M., Kobori, N.N., Jackson, M.A., Dunlap, C.A. and Delalibera Jr, I. 2018. Nitrogen sources affect productivity, desiccation tolerance, and storage stability of Beauveria bassiana blastospores. Journal of Applied Microbiology, 124, pp.810-820. https://doi.org/ 10.1111/jam.13694. Gandarilla-Pacheco, F. L., Morales-Ramos, L.H., Pereyra-Alférez, B., Elías-Santos, M. y Quintero-Zapata, I. 2018. Estudio comparativo de la producción de unidades infectivas de Isaria fumosorosea (Hypocreales: Cordycipitaceae) a partir de aislados nativos del noreste de México mediante tres estrategias de propagación. Revista Argentina de Microbiología, 50,pp. 81-89. https://doi.org/10.1016/j.ram.2017.03.008. Mascarin, G.M., Jackson, M.A., Kobori, N.N., Behle, R.W. and Delalibera Jr, I. 2015. Liquid culture fermentation for rapid production of desiccation tolerant blastospores of Beauveria bassiana and Isaria fumosorosea strains. Journal of Invertebrate Pathology, 127, pp. 11-20. https://doi.org/10.1016/j.jip.2014.12.001. Griffin, D.H.1996. Fungal physiology. 2da Ed. Estados Unidos: John Wiley & Sons, Inc. Hegedus, D.D., Bidochka, M.J. and Khachatourians G.G. 1990. Beauveria bassiana submerged conidia production in a defined medium containing chitin, two hexosamines or glucose. Applied Microbiology and Biotechnology, 33, pp. 641-647. https://doi.org/10.1007/BF00604930. Mishra, J., Tewari, S., Singh, S. and Arora, N.K. 2015. In: Arora, N.K. ed. Biopesticides: Where We Stand? Plant Microbes Symbiosis: Applied Facets. India: Springer. pp. 37-75. Pourseyed, S.H., Tavassoli, M., Bernousi, I. and Mardani K. 2010. Metarhizium anisopliae(Ascomycota: Hypocreales): An effective alternative to chemical acaricides against different developmental stages off owl tick Argaspersicus(Acari: Argasidae). Veterinary Parasitology, 172, pp. 305-310. https://doi.org/10.1016/j.vetpar.2010.05.014. Huarte‐Bonnet, C., Paixão, F.R.S., Mascarin, G.M., Santana, M., Fernandes, E.K.K. and Pedrini N. 2019. The entomopathogenic fungus Beauveria bassiana produces microsclerotia‐like pellets mediated by oxidative stress and peroxisome biogenesis. Enviromental Microbiology Reports, 11, pp.518-524. https://doi.org/10.1111/17582229.12742. Jackson, Rombach, M.C.1989. Production of Beauveria bassiana (Deuteromycotina. Hyphomycetes) sympodulo conidia in submerged culture. Entomophaga, 34, pp.45-52. https://doi.org/10.1007/BF02372586. M.A. 1997. Optimizing nutricional conditions for the liquid culture production of effective fungal biological control agents. Journal of Industrial Microbiology and Biotechnology, 19:180187.https://doi.org/10.1038/sj.jim.2900426. Sandoval-Coronado C.F., Quintero-Zapata I., Maldonado-Blanco M. G., Elías-Santos M., & Galán-Wong L. J. 2010. En: CoriaÁvalos, V. M., Lara-Chávez,M. B. N., Orozco-Gutiérrez, G., Muñoz-Flores, H. J., y Sánchez-Martínez, R. eds. Producción de Jackson, M.A., Cliquet, S., and Iten L.B. 2003. Media and fermentation processes for the rapid production of high concentrations of stable blastospores of the bioinsecticidal fungus 9 Tropical and Subtropical Agroecosystems 24 (2021): #80 Elías-Santos et al., 2021 blastosporas de Beauveria bassiana en un medio de cultivo líquido a base de glucosa y peptona de colágena. Memoria XXXIII Congreso Nacional de Control Biológico. Michoacán, México, INIFAP-CIRPAC. hongo-beauveria-bassiana-balsamovuillemin. Villarroel, P., Gómez, C., Vera, C., y Torres J. 2018.Almidón resistente: Características tecnológicas e intereses fisiológicos. Revista Chilena de Nutrición, 45, pp. 271-278. http://dx.doi.org/10.4067/s071775182018000400271. Villalba, P. L., Grillo-Ravelo, H., y Cupull, R. 2010. Evaluación de tres sistemas de agitación para la producción de blastosporas del hongo Beauveria bassiana (Bálsamo) Vuillemin, Centro Agrícola, 37, pp. 1721.http://cagricola.uclv.edu.cu/index.php/es/ volumen-37-2010/numero-1-2010/359evaluacion-de-tres-sistemas-de-agitacionpara-la-produccion-de-blastosporas-del- Zimmermann, G. 2007. Review on safety of the entomopathogenic fungi Beauveria bassiana and Beauveria brongniartii. Biocontrol Science and Technology, 17, pp. 553-596. https://doi.org/10.1080/09583150701309006. 10
W2914748593.txt	https://tumnig.tyuiu.ru/jour/article/download/667/661	en		PREDICTION OF PARAMETERS FOR COMMERCIAL OIL-AND-GAS CONTENT ZONES OF JURASSIC SEDIMENTS IN THE NORTH OF WESTERN SIBERIA	Izvestiâ vysših učebnyh zavedenij. Neftʹ i gaz	2,019	cc-by	3,020	Геология, поиски и разведка месторождений нефти и газа Geology, prospecting and exploration of oil and gas fields DOI: 10/31660.0445-0108-2018-6-9-15 УДК 553.98(571.12)+551.762 ПРОГНОЗ ПАРАМЕТРОВ ЗОН ПРОМЫШЛЕННОЙ НЕФТЕГАЗОНОСНОСТИ ЮРСКИХ ОТЛОЖЕНИЙ СЕВЕРА ЗАПАДНОЙ СИБИРИ Я. И. Гладышева Тюменский индустриальный университет, г. Тюмень, Россия Аннотация. Промышленная продуктивность юрских отложений на севере Западной Сибири подтверждена открытием залежей углеводородов на ряде месторождений. Большая часть их находится в среднеюрских отложениях, из нижнеюрских пластов получены лишь признаки углеводородов, из верхнеюрских — единичные залежи. Такое неравномерное распределение залежей в юрском нефтегазоносном объекте связано со сложностью его строения. Следовательно, необходимы комплексный анализ геологического строения юрских отложений, выявление геологических параметров для прогнозирования зон промышленной значимости на исследуемой территории. По результатам изучения данных глубокого бурения, сейсморазведочных работ севера Западной Сибири в юрском нефтегазоносном мегакомплексе построены схематические карты зон глубин кровли юрских отложений, определены параметры зон промышленной продуктивности. Наиболее значимым является наличие аномально высокого пластового давления. Исходя из проведенных исследований, отмечены предполагаемые зоны промышленной нефтегазоносности для юрских отложений в пределах Надым-Пурского междуречья. Ключевые слова: залежи углеводородов; глубокое бурение; коэффициент аномальности пластового давления PREDICTION OF PARAMETERS FOR COMMERCIAL OIL-AND-GAS CONTENT ZONES OF JURASSIC SEDIMENTS IN THE NORTH OF WESTERN SIBERIA Ya. I. Gladysheva Industrial University of Tyumen, Tyumen, Russia Abstract. Discovered hydrocarbon deposits in a number of fields are the characteristic of the industrial productivity of Jurassic sediments in the north of Western Siberia. Most of the deposits are in the Middle Jurassic sediments, the signs of hydrocarbons have been obtained in the Lower Jurassic sediments, single deposits have been obtained in the Upper Jurassic sediments. This uneven distribution of deposits in the Jurassic oil and gas bearing facility is associated with the complexity of its structure. Therefore, there is a need to conduct a comprehensive analysis of the geological structure of Jurassic sediments, to identify geological parameters to predict commercial oiland-gas content zones in the investigated territory. № 6, 2018 Нефть и газ 9 Based on the results of the study of deep drilling data, seismic survey in the north of Western Siberia, schematic maps of the depth zones of Jurassic sediments have been constructed, the author of the article determines the parameters of industrial productivity zones. The most significant parameter is abnormally high formation pressure. Based on the conducted researches, the author notes the prospective commercial oil-and-gas content zones for Jurassic sediments within NadymPur watershed. Key words: hydrocarbon deposits; deep drilling; formation pressure anomaly ratio Развитие ресурсной базы углеводородного сырья в нашей стране связано с поиском перспективных объектов углеводородов (УВ), в том числе и в юрских отложениях. Важной составляющей в изучении этого объекта является комплексный подход с использованием разных методов исследований. Юрский нефтегазоносный мегакомплекс подразделен на три комплекса: нижнеюрский, среднеюрский, верхнеюрский [1]. От нижезалегающих пород юрские отложения отделены поверхностью стратиграфического несогласия, от верхних отделены региональным репером, флюидоупором — аргиллитами баженовской свиты. Нижнеюрский нефтегазоносный комплекс на территории Надым-Пурского междуречья исследован единичными глубокими скважинами. На ВосточноУренгойской площади нижнеюрские породы вскрыты сверхглубокой скв. СГ6 на глубине 4 710 м, на Уренгойской площади — глубокой скв. 414 (гл. 4 321 м), на Ен-Яхинской площади — сверхглубокой скв. СГ7 (гл. 4 810 м), на Юбилейной площади — глубокой скв. 200 (гл. 4 386 м), на Медвежьей площади — скв. 1 001 (гл. 3 805 м), на Надымской площади — скв. 7 (гл. 4 000 м), на Ямсовейской площади — скв. 98 (гл. 3 762 м). Глубина кровли нижнеюрских отложений на большей части исследуемой территории находится в диапазоне от 3,0 до 5,0 км. Минимальная глубина залегания нижнеюрских отложений установлена в южной части исследуемой территории на Ямсовейской площади в скв. 98 (3 762 м), максимальная глубина — на северо-востоке Ен-Яхинской площади в скв. СГ7 (4 810 м). Перепад глубин кровли нижнеюрских отложений составил около 1 148 метров. Толщина нижнеюрских отложений изменяется от 462 м на югозападе (Надымская площадь, скв. 7) до 1 302 м на востоке (Восточно-Уренгойская площадь, скв. СГ6) и 1 340 м на северо-востоке (Ен-Яхинская площадь, скв. СГ7), разница максимальных и минимальных общих толщин составляет 878 метров. По данным бурения и интерпретации сейсморазведочных работ автором статьи была построена схематическая карта зон глубин кровли нижнеюрских отложений севера Западной Сибири. По современным глубинам кровля нижнеюрских отложений представляет собой сложную расчлененную поверхность, чередование более приподнятых блоков с опущенными, при общей тенденции погружения с запада на северо-восток. Значительное изменение общей толщины нижнеюрских отложений подтверждает сложное блоковое строение изучаемой территории, где для каждого блока характерен свой тектогенез [2]. Увеличение толщины нижнеюрских отложений в северо-восточной части изучаемой территории обусловлено большей амплитудой опускания и близостью источника сноса, где скорость накопления осадков была выше, чем в западной части. Отложения нижнеюрского нефтегазоносного комплекса на изучаемой территории Надым-Пурского междуречья представлены неравномерным переслаиванием песчаников, алевролитов и аргиллитов, в нижней части разреза встречаются прослои гравелитов и конгломератов. В нижнеюрском разрезе толщина пропластков коллекторов изменяется от 0,4 до 4,8 м и в среднем составляет 1,2 м, в разрезе преобладают глинистые разности. Пласты-коллекторы характеризуются значи- 10 Нефть и газ № 6, 2018 тельной неоднородностью и не выдержаны. Фильтрационно-емкостные параметры низкие, пористость изменяется от 10 до 16 % и в среднем равна 13 %, проницаемость изменяется от 1∙10-3 до 20∙10-3 мкм2. В большинстве скважин при испытании нижнеюрских отложений выявлены лишь признаки УВ. Исключение — Медвежье месторождение, где в скв. 1 001 из пласта Ю13 получен приток газа. Отсутствие промышленных притоков флюидов из нижнеюрских отложений связано с низкими фильтрационно-емкостными свойствами (ФЕС) коллекторов, их значительной глинизацией и качеством вскрытия пород. Последний фактор значительно влияет на изучение глубокозалегающих горизонтов. Пластовая температура нижнеюрского комплекса изменяется от 100 до 120 0С. Общая минерализация пластовой воды на территории Надым-Пурского междуречья составила в среднем 16,0 г/л, тогда как на полуострове Ямал она в среднем около 12,5 г/л. В нижнеюрских отложениях залежи УВ открыты на полуострове Ямал (Бованенковское и Новопортовское месторождения). На этих месторождениях отложения вскрыты на глубинах от 2 490 до 3 130 м, общая толщина их составляет около 450 м, коэффициент аномальности пластового давления в среднем 1,5. С учетом фактических данных по нижнеюрскому нефтегазоносному комплексу на изучаемой территории отмечены определенные закономерности. Промышленная продуктивность выявлена при наличии основных факторов: первый фактор — приуроченность к антиклинальной структуре (глубина залегания в пределах от 2 000 до 3 000 м) со значительной амплитудой (по отражающему сейсмическому горизонту Т4 более 200 м); второй фактор — литологический состав нижнеюрских отложений (наличие эффективных коллекторов преимущественно поровотрещинного типа и надежных покрышек); третий фактор — аномальное пластовое давление, где коэффициент аномальности пластового давления более 1,5. В пределах Надым-Пурского междуречья с большей долей вероятности прогнозируются промышленные скопления УВ в нижней юре в юго-западной части изучаемой территории. Район территории Ямала (Бованенковское и Новопортовское месторождения) автор связывает с тектонически активной зоной. К северовостоку наблюдается погружение нижнеюрских отложений, где возможны поиски сложных структурно-литологических и тектонически экранированных ловушек УВ. Среднеюрские отложения (тюменская свита) изучены бурением и сейсморазведочными работами на большей части Надым-Пурской территории, но неравномерно. Вскрыта в основном верхняя часть разреза — пласты Ю2–Ю3. С учетом материалов глубокого бурения и интерпретации сейсморазведочных работ также была построена схематическая карта зон глубин кровли среднеюрских отложений севера Западной Сибири. Минимальное значение глубины кровли тюменской свиты (3 108 м) фиксируется на юге исследуемой территории на Ямсовейско-Юбилейном валу (скв. 98 Ямсовей) и на западе — на Медвежьей структуре (скв. 31 Медвежья). На востоке — на Уренгойской площади кровля тюменской свиты установлена на глубинах от 3 600 до 3 850 м, севернее — на Песцовой и Ен-Яхинской площадях диапазон глубин составляет 3 800–3 950 м. Между Медвежьим и Уренгойским мегавалами расположена Нерутинская впадина (структура I порядка), где глубина кровли тюменской свиты варьирует в пределах 3 900–4 200 м, амплитуда — около 300 метров [3]. Максимальное значение глубины залегания кровли тюменской свиты отмечается на севере изучаемой территории на Ямбургской структуре — 4 314 м (скв. 183 Ямбургская). Перепад глубин кровли тюменской свиты с юга на север составляет около 1,2 км. Амплитуды положительных структур второго порядка Медвежьего и Ямсовейского валов составляют в среднем около 200 метров. Тогда как для Уренгойского мегавала амплитуда в среднем около 100 м, для Ямбургского вала — около 60 м. Общая мощность тюменской свиты по данным скважин изменяется № 6, 2018 Нефть и газ 11 от 400 м на северо-западе (скв. 7 Новопортовская) и 520 м на юго-западе (скв. 7 Надымская) до 860 м на севере (скв. СГ7 Ен-Яхинская). Как и по нижнеюрским отложениям, в среднеюрском разрезе сохраняется тенденция увеличения мощности отложений с запада на северо-восток, с увеличением глубины залегания кровли тюменской свиты увеличивается и общая толщина. Однако для среднеюрских отложений разница между минимальными и максимальными толщинами варьирует в пределах 30 %, тогда как для нижнеюрских отложений это 50–70 %. При сравнении схематических карт зон глубин кровли нижнеюрских и среднеюрских отложений было выявлено, что основные структурные элементы I и II порядка развивались унаследовано. Медвежий и Ямсовейско-Юбилейный крупные валы приурочены к наиболее приподнятым блокам. Контраст их по сравнению с Уренгойским мегавалом (структура I порядка) в нижне- и среднеюрский периоды составил по современным глубинам около 500 метров. Геологический разрез тюменской свиты представлен неравномерным переслаиванием песчаников, алевролитов и аргиллитов с преобладанием последних. В керне отмечены мелкие стяжения пирита, встречаются сидерит, глауконит, обрывки углефицированной флоры, ходы донных животных, многочисленные прослои угля, достигающие толщины 90 см. В среднеюрском разрезе толщина пропластков коллекторов изменяется от 0,4 до 12,0 м и в среднем составляет 1,4 м, в разрезе преобладают глинистые разности. Фильтрационно-емкостные параметры в большинстве низкие, пористость изменяется от 11 до 20 % и в среднем около 15 %, проницаемость — от 1∙10-3 мкм2 до 60∙10-3 мкм2. Необходимо отметить, что промышленные притоки УВ из среднеюрских отложений получены из коллекторов различных фаций, в том числе из пород с низкими ФЕС. Пластовое давление в залежах изменяется от 31 до 80 МПа и в среднем составляет 70 МПа. Коэффициент аномальности пластового давления изменяется от 1,4 до 2,1 и в среднем равен 1,9. Пластовая температура изменяется от 88 до 120 0С [4]. Автором отмечено, что промышленные притоки УВ залежей пластов Ю2–Ю3 получены там, где коэффициент аномальности пластового давления более 1,8. В диапазоне, где коэффициент аномальности составляет 1,7–1,8, притоки УВ значительно меньше. В зонах, где коэффициент аномальности пластового давления менее 1,6, на исследуемой территории по результатам бурения были получены лишь признаки УВ. Анализ термобарических параметров позволил выделить на исследуемой территории Надым-Пурского междуречья три зоны с максимальными значениями коэффициента аномальности пластового давления. Основная зона аномальности, где коэффициент аномальности превышает 1,9, приурочена к северной и центральной частям территории (Песцовая, Ен-Яхинская, Западно-Песцовая, Ямбургская площади), где открыты залежи УВ в пластах Ю2–Ю3. Зона коэффициента аномальности более 1,7 охватывает Юбилейное и Уренгойское месторождения на юго-востоке, на северо-западе — Северо-Парусовое. Между коэффициентом продуктивности (отношение дебита УВ к депрессии) и коэффициентом аномальности пластового давления отмечена связь, где коэффициент продуктивности возрастает при значениях коэффициента аномальности более 1,9 (рисунок). В среднеюрских отложениях на исследуемой территории залежи УВ открыты на Уренгойском, Песцовом, Ямбургском, Юбилейном и Восточно-Медвежьем месторождениях. Максимальный этаж нефтегазоносности в среднеюрском разрезе выявлен на Уренгойском месторождении (открыто около семнадцати залежей УВ 12 Нефть и газ № 6, 2018 в пластах Ю2–Ю4), по насыщению залежи преимущественно нефтяные. Тогда как на Песцовом и Ямбургском месторождениях залежи пласта Ю2 газовые, на Юбилейном и Восточно-Медвежьем — газоконденсатно-нефтяные. Рисунок. Зависимость коэффициента аномальности от коэффициента продуктивности среднеюрских залежей углеводородов севера Западной Сибири Верхнеюрские отложения (баженовская и абалакская свиты) исследованы более детально сейсморазведочными работами и бурением. С учетом данных интерпретации сейсморазведочных работ, материалов бурения, структурных карт В. А. Галунского по отражающему горизонту Б автором статьи построена схематическая карта кровли глубин баженовской свиты, где центральную часть изучаемой территории занимает структура I порядка — Нерутинская впадина, максимальная глубина которой фиксируется на Падинской площади — 4 036 м (скв. 5 400). Большая часть Нерутинской впадины оконтуривается изогипсой глубины кровли баженовской свиты 4 000 метров. С запада Нерутинская впадина граничит со структурой II порядка — Медвежьим валом, который по изогипсе глубины 3 300 м разделяется на два купола. На севере — это Ныдинское поднятие, а в центральной и южной частях — Медвежье куполовидное поднятие. На юге последнего фиксируется минимальная глубина залегания кровли баженовских отложений на Медвежьей площади — 3 069 м (скв. 31). Контраст современных глубин кровли баженовской свиты между Медвежьим валом и Нерутинской впадиной — около 500 метров. Наименьшее значение глубины кровли баженовской свиты на исследуемой территории отмечается на Ямсовейско-Юбилейном крупном валу (структура II порядка), в центральной части Ямсовейского вала — 3 033 м (скв. 98). По изогипсе глубины кровли баженовской свиты 3 700 м Ямсовейский вал на северо-востоке переходит в Юбилейное куполовидное поднятие, где минимальная глубина кровли баженовской свиты составляет 3 388 м (скв. 1 001). По изогипсе глубины кровли баженовских отложений 3 600 м на Ямсовейском валу амплитуда составила 567 м, тогда как на Юбилейной структуре амплитуда — около 210 метров. По современным глубинам кровли баженовской свиты наибольшие значения амплитуд антиклинальных поднятий связаны с юго-западной частью изучаемой территории (центр Ямсовейского вала и юг Медвежьего локального поднятия). С востока Нерутинская впадина граничит со структурой I порядка — Уренгойским мегавалом, который по изогипсе глубины 3 600 м разделяется на три купола (Северо-Уренгойское, Центрально-Уренгойское I, Центрально-Уренгойское II куполовидные поднятия). По замыкающей изогипсе глубины 3 800 м амплитуды ло- № 6, 2018 Нефть и газ 13 кальных поднятий Уренгойского мегавала составляют около 200 метров. Контраст глубин кровли баженовской свиты между Уренгойским мегавалом и Нерутинской впадиной около 200 метров. На севере Нерутинской впадины расположены Песцовое и Ен-Яхинское куполовидные поднятия, которые по изогипсе глубины кровли баженовской свиты 3 900 м объединены в одну антиклинальную структуру. На Песцовом поднятии минимальная глубина залегания кровли бажена составляет 3 724 м (скв. 208), на Ен-Яхинском — 3 744 м (скв. 610). Амплитуды этих локальных поднятий по изогипсе глубин 3 800 м — около 60–80 м. Такие же значения амплитуды фиксируются на Харвутинском купловидном поднятии, где минимальная глубина кровли баженовских отложений — 3 740 м (скв. 253). Севернее, на Ямбургском куполовидном поднятии амплитуда по изогипсе кровли 3 800 м составила около 200 метров. Максимальная глубина кровли баженовских отложений фиксируется на северовостоке изучаемой территории — 4 177 м (скв. 102 Северо-Самбургская). Анализ современных глубин кровли баженовских отложений показал, что значения глубин на исследуемой территории варьируют в диапазоне от 3,0 до 4,2 км, с общей тенденцией погружения на северо-востоке. По схематической карте общей толщины баженовских отложений максимальные значения (более 60 м) фиксируются на северо-западе исследуемой территории в пределах Ныдинского локального поднятия Медвежьего вала. Изопахита 50 м охватывает как Ныдинский участок, так и соседний Южно-Падинский. На северовостоке в скв. СГ7 Ен-Яхинской площади мощность баженовских отложений составляет 52 метра. На Песцовом и Ен-Яхинском куполовидных поднятиях общая толщина бажена изменяется в диапазоне от 15 до 28 м, в среднем — около 19 метров. На севере в пределах Ямбургского куполовидного поднятия общая толщина баженовской свиты изменяется от 26 (скв. 503) до 36 м (скв. 183), в среднем — 30 метров. Такие же значения общей толщины отмечены в пределах большей части Нерутинской впадины, на юге Медвежьего вала и в пределах Юбилейного поднятия. На Уренгойской площади значения общей толщины баженовских отложений характеризуются наименьшими величинами и изменяются от 8 (скв. 805) до 20 м (скв. 414), в среднем — около 15 метров. В пределах Ямсовейского вала общая толщина баженовских отложений выдержана по площади и разрезу, в среднем составляет 17 метров. Тогда как на соседнем ЗападноЯгенетском участке, прилегающем на востоке, в скв. 10 отмечено увеличение общей толщины баженовской свиты до 42 метров. Анализируя схематическую карту общей толщины баженовских отложений, можно отметить, что большую часть Надым-Пурского междуречья занимают зоны общих толщин от 10 до 30 м. Зоны общих толщин более 30 м расположены локально, на отдельных участках. Максимальные значения общей толщины баженовских отложений более 50 м, как отмечено выше, расположены на северо-западе. Значительное увеличение общей толщины бажена ряд ученых называют «аномальным баженом». Генезис такого разреза связывают с опесчаниванием разреза за счет внедрения песчанистого материала в битуминозные глинистые осадки при позднеюрских и раннемеловых тектонических движениях [3]. Формирование баженовских отложений на изучаемой территории проходило в глубоководном бассейне, при относительно выравненном палеорельефе, но с различной скоростью накопления на разных участках. Большие мощности палеоосадков накапливались на пониженных участках, меньшие — на приподнятых. По верхнеюрским отложениям автором была построена схематическая карта общей толщины, где параметр общей толщины изменяется в диапазоне от 31 (скв. 5 011 Медвежья) до 186 м (скв. 183 Ямбургская) и в среднем составляет 94 метра. 14 Нефть и газ № 6, 2018 Максимальные значения общей толщины верхнеюрских отложений более 130 м отмечены на отдельных участках. Больший по площади участок картируется на севере изучаемой территории в пределах Ямбургской и Южно-Парусовой площадей. На Падинском, СевероСамбургском и Западно-Ягенетском участках зоны максимальных толщин имеют локальное распространение в пределах отдельных скважин. Минимальные значения общей толщины фиксируются в центральной и восточной частях Медвежьей площади. Северная часть Нерутинской впадины характеризуется толщинами в пределах 90–110 м, в южной ее части — 70–90 м. На территории Уренгойского мегавала отмечается тенденция увеличения общей толщины в северной части по сравнению с южной на 20–30 метров. На момент, когда сформировались верхнеюрские отложения на Медвежьей и Уренгойской площадях, южные части этих структур были более приподняты относительно северных частей, где формировались осадки меньшей толщины, а опущенные зоны-блоки располагались на Ямбургском, Южно-Парусовом, между Ныдинским и Песцовым участками, на Северо-Самбургском и Западно-Ягенетском участках. Верхнеюрские отложения по литологическому составу представлены аргиллитами с редкими прослоями алевролитов, реже песчаников. На исследуемой территории из этих отложений получены непромышленные притоки углеводородов. Только на Западно-Юбилейном участке из отложений абалакской свиты пласта Ю10 получен фонтанный приток нефти. Южнее изучаемой территории, на площадях, где развиты отложения васюганской свиты (горизонт Ю1), открыты залежи углеводородов, газовая залежь — на Северо-Губкинском месторождении, нефтяные залежи на Яранейрском, Суторминском, Губкинском, Етыпурском, Апакопурском, Новогоднем, Вынгапурском и Вынгаяхинском месторождениях. То есть для верхнеюрских отложений важным параметром является наличие прослоев коллектора, поиск которых возможен с применением 3D-сейсморазведочных работ. А для нижезалегающих юрских отложений важным параметром является коэффициент аномальности пластового давления. Библиографический список 1. Тектоническое строение и история развития Западно-Сибирской геосиниклизы в мезозое и кайнозое / В. А. Конторович [и др.] // Геология и геофизика. – 2001. – № 11–12. – С. 1832–1845. 2. Геологическое строение и нефтегазоносность нижней-средней юры Западно-Сибирской провинции / Ф. Г. Гурари [и др.]. – Новосибирск: Наука, 2005. – 197 с. 3. Нежданов А. А., Скрылев С. А., Огибенин В. В. Нерутинская впадина — новый нефтегазоносный район Западной Сибири. Перспективы и проблемы // Материалы XIV координационного геологического совещания. – М.: ООО «Газпром экспо», 2009. – С. 14–29. 4. Гладышева Я. И. Проблемы поисковых работ севера Западной Сибири // Нефтепромысловое дело. – 2009. – № 8. – С. 21–32. Сведения об авторе Гладышева Яна Игоревна, к. г.-м. н., доцент кафедры геологии месторождений нефти и газа, Тюменский индустриальный университет, г. Тюмень, e-mail: gladyshevaji@tyuiu.ru № 6, 2018 Information about the author Gladysheva Ya. I., Candidate of Geology and Mineralogy, Associate Professor at the Department of Geology of Oil and Gas Fields, Industrial University of Tyumen, e-mail: gladyshevaji@tyuiu.ru Нефть и газ 15
https://openalex.org/W2192090293	https://revistes.ub.edu/index.php/oximora/article/download/14472/17855	Spanish; Castilian	null	Feminismo como crítica: Sujeto y universalidad	DOAJ (DOAJ: Directory of Open Access Journals)	2,015	cc-by	5,096	Abstract: Firstly, this article aims to refute some approaches of ´difference feminism` using as a guide Chantal Mouffe and Judith Butler proposals, both linked to the defense of a women open concept. Later, also resorting to populist proposal of Ernesto Laclau, we’ll argue in favor of a political feminism understood as a possible tool of unification of various struggles for the construction of a counter-hegemony. Resumen: En primer lugar, el presente artículo pretende refutar algunos planteamientos propios del «feminismo de la diferencia» utilizando como línea argumental las propuestas de Chantal Mouffe y Judith Butler, ligadas en la defensa de un concepto abierto de mujer. Posteriormente, recurriendo también a la propuesta populista de Ernesto Laclau, se argumentará en favor de un feminismo político entendido como una posible herramienta de unificación de luchas diversas por la construcción de una contra- hegemonía. Palabras clave: Contingencia, significante, demanda, hegemonía. FEMINISMO COMO CRÍTICA: SUJETO Y UNIVERSALIDAD Carlos Villanueva Castro Universidad de Valencia Carlos Villanueva Castro INTRODUCCIÓN Desde diversos ámbitos de debate y reflexión se ha venido incidiendo en la idea fundamental de que el feminismo es algo que trasciende sus propios orígenes como movimiento político y que en el campo filosófico juega un rol importantísimo. El feminismo puede entenderse no sólo como ese conjunto de propuestas y teorías que, con alianzas o enfrentamientos, han acompañado a la lucha de las mujeres por su liberación, sino que, en el terreno teórico así como en el práctico, el feminismo ofrece la oportunidad de repensar lo dado y trascender el presente. El feminismo filosófico, atendiendo a cuestiones de su propia índole, puede alcanzar respuestas o resituar los horizontes de sentido de un modo que afecta a otros ámbitos del pensamiento. Por ello hablamos de feminismo como crítica, como un análisis reflexivo que trasciende su propia idiosincrasia para vincularse a cuestiones políticas, ontológicas o antropológicas que implican al conjunto de terrenos teóricos por los que los amantes de las preguntas transitan. En este ensayo trataremos de apuntar hacia las posibilidades, incluso la necesidad, de que el feminismo teórico, atendiendo a problemas ya clásicos de su propia disciplina pueda trascenderla y aportar ideas al conjunto de la reflexión filosófica. En primer lugar atenderemos a la pregunta por el quién del feminismo, un interminable debate que conlleva a escoger por una respuesta esencialista o una vinculada a la contingencia. ¿Quién es el sujeto del que trata el feminismo? ¿Qué significa ser mujer? ¿Qué cabe dentro de tal término y bajo qué condiciones? Recurriendo a un artículo de Chantal Mouffe y a diversos textos de Judith Butler pondremos en cuestión la idea de un feminismo de la diferencia como única alternativa al androcentrismo, y después, la necesidad de un sujeto lábil y voluble que no reifique la identidad de la mujer precisamente en su lucha por la emancipación. A continuación estableceremos los vínculos que existen entre las propuestas de Mouffe, Butler y Ernesto Laclau en lo referente al carácter de este sujeto político del feminismo y sus posibilidades emancipatorias más allá de su parcialidad dentro del terreno de las diferentes luchas y movimientos sociales. 1 Principalmente en su famosa obra La razón populista (2005). Key words: Contingency, significant, demand, hegemony. Recibido: 28/10/2015 Aceptado: 15/11/2015 OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 203 Feminismo como crítica: sujeto y universalidad OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 INTRODUCCIÓN El feminismo puede contener en su seno diferentes formas de identificación y de luchas del mismo modo que el feminismo puede formar parte de un conjunto mayor de enfrentamientos por un nuevo orden e incluso representar, al modo en que lo propone Laclau cuando habla de significantes vacíos1, el conjunto de luchas con las que se da un lazo equivalencial. Esta posibilidad no implica rechazar los rasgos propios y distintivos del conjunto de posicionamientos feministas y de prácticas liberadoras de la opresión machista o patriarcal, sino la posibilidad de que en esas luchas se dé el reflejo de otras tantas y viceversa. OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 204 Carlos Villanueva Castro El motivo de la elección de esta temática responde a diversas inquietudes de quien escribe. La preocupación por las propuestas del feminismo de la diferencia, considerando que pueden incurrir fácilmente en uno de los elementos, el esencialismo y el cierre identitario, que forman parte de aquello contra lo que el feminismo ha venido oponiéndose en sus luchas políticas, era uno de los ingredientes que se pretendía introducir en este texto. Por otra parte, la necesidad de que el feminismo forme parte de la lucha, junto a otras tantas, por la construcción de una nueva hegemonía social y en el auge de nuevos valores y perspectivas, todavía no suficientemente arraigados, era otra de las preocupaciones latentes. La lectura de Mouffe y Laclau permite encontrar numerosos parecidos con muchas de las ideas clave de Butler, de modo que resultó interesante enlazarlas precisamente en la dirección de estas inquietudes teóricas, y prácticas, en este ensayo. La conclusión, que ya podemos adelantar, es que el feminismo no puede referirse a un sujeto político previo a la propia praxis ni a un sujeto constituido pero finiquitado del que ya no cabe cuestionamiento alguno. Pero precisamente esta contingencia radical en la que reposa toda identidad política es la que permite que se establezcan vínculos de toda índole entre las diferentes luchas sociales y la que posibilita que estas luchas unidas configuren nuevas formas de acción y pensamiento que se enfrenten al estado de cosas vigente. En la configuración de esa amalgama de luchas el feminismo puede y debe jugar un papel protagonista. EL SUJETO DEL FEMINISMO En el artículo “Feminismo, ciudadanía y política democrática radical” (1996), Chantal Mouffe apuesta por la deconstrucción de las identidades esenciales asumiendo la contingencia como base de toda constitución de sujeto. No hay nunca un sujeto previo a la acción, sino que hay un cúmulo de acciones y de actos que configuran un sujeto. Sin embargo para muchas feministas el rechazo de una noción fuerte de mujer en la que sustentar la lucha feminista supone el abandono de su particular proyecto emancipatorio. Mouffe en este artículo trata de deslegitimar el conjunto de posturas que apelan a una identidad homogénea y concreta de mujer como elemento indispensable de la reflexión y lucha feminista, afirmando precisamente lo contrario, que bajo el modelo de democracia radical que ella defiende no cabe ningún principio esencialista que impida la efectividad de la pluralidad propia de las sociedades modernas. Que el sujeto no sea algo dado, sino el resultado de diversas posiciones de sujeto o cristalizaciones temporales y precarias de movimientos identitarios no supone rechazar el mismo concepto de mujer en la lucha feminista, pero mucho menos implica abocarlo a una definición única y cerrada. OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 205 Feminismo como crítica: sujeto y universalidad Este planteamiento viene a cuestionar la disputa entre el feminismo de la diferencia y el feminismo de la igualdad, pues la polémica sólo es posible si se asume que la igualdad o la diferencia están sustentadas en una identidad firme y cerrada, homogénea e invariable: Este planteamiento viene a cuestionar la disputa entre el feminismo de la diferencia y el feminismo de la igualdad, pues la polémica sólo es posible si se asume que la igualdad o la diferencia están sustentadas en una identidad firme y cerrada, homogénea e invariable: «Todo el falso dilema de la igualdad versus la diferencia se derrumba desde el punto en que ya no tenemos una entidad homogénea “mujer” enfrentada con otra entidad homogénea “varón”, sino una multiplicidad de relaciones sociales en las cuales la diferencia sexual está construida siempre de muy diversos modelos y donde la lucha en contra de la subordinación tiene que ser planteada en formas específicas y diferenciales» (Mouffe, 1996: 6). EL SUJETO DEL FEMINISMO Mouffe emplea parte de su artículo para cuestionar propuestas concretas de feminismo de la diferencia, como son las de Carol Gilligan apelando a una ética del cuidado propia de las mujeres o Sara Ruddick y Jean Bethke defendiendo la idea de un pensamiento maternal configurado a partir de la experiencia de las mujeres como madres dentro del ámbito privado y de la familia. Estas propuestas apuntan a la asunción de unos rasgos específicos propios de la mujer o de la esfera privada a la que han sido relegadas las mujeres para visibilizarlos y enfatizarlos frente a los elementos masculinos que ocupan lo público en la constitución de una nueva ciudadanía. Pero como apunta Mouffe basándose en la crítica de Mary Dietz a estas propuestas, no puede constituirse una concepción de lo público desde posiciones del tipo de las relaciones materno-filiales en las que media un vínculo afectivo que difícilmente puede reproducirse en todos los ámbitos y con todas las personas involucradas en la convivencia cívica. Por otro lado Mouffe cita a Carole Pateman y su propuesta de una concepción sexualmente diferenciada de la ciudadanía, defendiendo una experiencia propia de las mujeres que se debe asumir para no caer en la opresión de la experiencia masculina. Si a la ciudadanía en general se le atribuye como virtud suprema la voluntad de pelear y morir por la patria, propia del varón, la capacidad de crear vida, definitoria de la maternidad, debe ser el elemento femenino que se distinga en la forma de configurar las mujeres su ciudadanía. Pateman, queriendo huir del androcentrismo que impone modelos de ciudadanía que han excluido a las mujeres y a los valores asociados a ellas del ámbito público, acaba aceptando la reificación y diferenciación sexual que conlleva ese androcentrismo y que parece impedir que la masculinidad o feminidad se vivan de modos distintos a los imperantes. Mouffe apuesta justo por lo contrario: OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 206 Carlos Villanueva Castro «No hay razón para que la diferencia sexual tenga que ser pertinente en todas las relaciones sociales. Desde luego, hoy en día existen muchas prácticas, discursos e instituciones diferentes que construyen (diferencialmente) a los varones y a las mujeres, y la distinción masculino/femenino existe como una distinción pertinente en muchos campos. EL SUJETO DEL FEMINISMO Pero esto no quiere decir que así tenga que seguir siendo, y nos podemos imaginar perfectamente bien la posibilidad de que la diferencia sexual se convierta en algo irrelevante en muchas de las relaciones sociales en que actualmente es relevante. De hecho, éste es el objetivo de muchas de las luchas feminista» (Mouffe, 1996: 12). La idea de unos valores o rasgos femeninos, que en muchas propuestas, como las que hemos mencionado, apelan a la maternidad, o en otros, al acercamiento con la naturaleza, frente a la racionalidad instrumental y destructiva de la masculinidad, continúan en la estela reificadora y sustancialista que en tiempos pasados, y también presentes, identifican a la mujer con unos roles concretos en los que el hombre mantiene una superioridad o incluso la propiedad sobre ella. La solución no es sustituir un sustancialismo por otro y donde antes los valores masculinos primaban situar ahora a los femeninos, lo que el feminismo crítico debiera intentar es luchar contra la opresión de las mujeres también enfrentándose a la homogeneidad y a la limitación de la propia identidad. Estos roles de género o estas identificaciones deterministas pueden ser tan opresivas y limitantes como las anteriores lo eran para las mujeres y también para los hombres que no cumplían con los modelos prefijados. ¿Es posible mantener la lucha feminista sin un sujeto conciso y claro y sin un concepto compartido de mujer? Para Mouffe no sólo es posible, es necesario: «La ausencia de una identidad esencial femenina y de una unidad previa, sin embargo, no impide la construcción de múltiples formas de unidad y de acción común. Como resultado de la creación de puntos nodales, pueden tener fijaciones parciales y pueden establecer formas precarias de identificación alrededor de la categoría de “mujeres”, que provean la base para una identidad feminista y una lucha feminista» (Mouffe, 1996: 18). El feminismo debe articularse en torno a los distintos modos de pensamiento feminista y a las diversas maneras de entender la categoría de mujer, sin que las muchas diferencias supongan un problema siempre que las similitudes permitan un vínculo equivalencial que sitúe a todos los discursos y prácticas en un mismo eje. No hay, pues, un verdadero modo de ser feminista ni una esencia irrevocable de la mujer. OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. Feminismo como crítica: sujeto y universalidad Feminismo como crítica: sujeto y universalidad En “Actos performativos y constitución del género: un ensayo sobre fenomenología y teoría feminista”(1998) Butler expone que tanto G. Spivak como Kristeva proponen un uso estratégico de la categoría mujeres, como herramienta política sin dotarle de carácter ontológico, pues estrictamente hablando, parafraseando a Kristeva, es dudoso incluso que las mujeres existan: «Pero una cosa es utilizar el término y conocer su insuficiencia ontológica, y otra cosa muy distinta es, para la teoría feminista, articular una visión normativa que celebre o emancipe una esencia, una naturaleza, o una realidad cultural compartida imposible de encontrar» (Butler, 1998:312). Las posturas feministas que consideran necesario para la liberación la defensa de un modelo binario de diferenciación sexual acaban más encadenadas debido a que reproducen el camino de la cosificación y la ontologización: «Mi única preocupación es que la diferencia sexual no se vuelva una cosificación que involuntariamente preserve una restricción binaria de la identidad de género y un marco implícitamente heterosexual para la descripción del género, la identidad de género y la sexualidad» (Butler, 1998: 314). Butler propone en este artículo que el género es la suma del conjunto de actos performativos que lo constituyen, no hay, pues, un sexo dado al que el género debe amoldarse, ni una identidad de género prefijada en la que cada gesto o acto del cuerpo debe inscribirse. Antes bien, cada gesto y acto configuran una suma de solidificaciones o puntos nodales de los que puede afirmarse que constituyen una identidad de género. El género nunca es correcto o incorrecto, a no ser que se siga la lógica binaria dominante según la cual hay un modelo ideal de género a seguir correspondiendo a cada sexo. Precisamente Butler, en El género en disputa (2007), propone el uso del método genealógico como herramienta para señalar el origen de este tipo de categorías de identidad que son efecto de instituciones, prácticas y razonamientos cuya historia se ha desdibujado haciendo pasar a determinados modelos de conducta como naturales o necesarios. Desde Marx, nos dice Butler, el principio de la filosofía de la sospecha ha sido el de la crítica cultural y la desconfianza frente a lo que se presenta como absoluto e ineludible, la reflexión en torno a los procesos de reificación. Las conclusiones de la obra son precisamente que el sujeto es una construcción OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. EL SUJETO DEL FEMINISMO OTOÑO 2015. ISSN 2014-7708. PP. 202-213 207 Feminismo como crítica: sujeto y universalidad ISSN 2014-7708. PP. 202-213 OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 208 Carlos Villanueva Castro de prácticas por repetición, de nuevo, la idea de que no hay un sujeto previo a la acción. El feminismo debe abandonar todo residuo de esencialismo y abrazar el postulado nietzscheano de que no hay un sujeto dado tras el predicado, todo es un hacer y el agente es añadido ficticiamente a posteriori. En este sentido, el feminismo se equivoca si apuesta por un sujeto metafísico no contextualizado. Por el contrario, Butler propone lo paródico como vía de subversión de los modelos y roles hegemónicos, precisamente para hacer ver su contingencia: «Al imitar el género, la travestida manifiesta de forma implícita la estructura imitativa del género en sí, así como su contingencia. En realidad, parte del placer, la frivolidad de la actuación, reside en la aceptación de una contingencia radical en la relación entre sexo y género frente a configuraciones culturales de unidades causales que suelen verse como naturales y necesarias. En vez de la ley de coherencia heterosexual vemos el sexo y el género desnaturalizados mediante una actuación que asume su carácter diferente y dramatiza el mecanismo cultural de su unidad inventada» (Butler, 2007: 269). En la parte inicial de la obra Butler aborda, precisamente, la cuestión que Mouffe trata en su artículo, la relevancia del concepto de mujer en la lucha feminista y los problemas que acarrea rechazar o asumir un concepto de mujer cerrado: «Para la teoría feminista, el desarrollo de un lenguaje que represente de manera adecuada y completa a las mujeres ha sido necesario para promover su visibilidad política. Evidentemente, esto ha sido de gran importancia, teniendo en cuenta la situación cultural subsistente, en la que la vida de las mujeres se representaba inadecuadamente o no se representaba en absoluto. «Para la teoría feminista, el desarrollo de un lenguaje que represente de manera adecuada y completa a las mujeres ha sido necesario para promover su visibilidad política. Evidentemente, esto ha sido de gran importancia, teniendo en cuenta la situación cultural subsistente, en la que la vida de las mujeres se representaba inadecuadamente o no se representaba en absoluto. Recientemente, esa concepción dominante sobre la relación entre teoría feminista y política se ha puesto en tela de juicio desde dentro del discurso feminista. Feminismo como crítica: sujeto y universalidad El tema de las mujeres ya no se ve en términos estables o constantes.(…) En efecto, la cuestión de las mujeres como sujeto del feminismo plantea la posibilidad de que no haya un sujeto que exista “antes” de la ley, esperando la representación en y por esta ley. Quizás el sujeto y la invocación de un “antes” temporal sean creados por la ley como un fundamento ficticio de su propia afirmación de legitimidad. La hipótesis prevaleciente de la integridad ontológica del sujeto antes de la ley debe ser entendida como el vestigio contemporáneo de la hipótesis del estado de naturaleza, es fábula fundacionalista que sienta las bases de las estructuras jurídicas del liberalismo clásico» (Butler, OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 209 Feminismo como crítica: sujeto y universalidad 2007: 46-48). Al no ser una noción estable de género la premisa principal de la política feminista, Butler apuesta por combatir las reificaciones mismas de género e identidad, sosteniendo «…que la construcción variable de la identidad es un requisito metodológico y normativo, además de una meta política.» (Butler, 2007: 53) Ya en el artículo “Fundamentos contingentes” (2001) Butler apostaba por un concepto de mujer abierto a múltiples significaciones frente al previsible fracaso de la imposición de un contenido universal y uniformador del mismo que conduciría a la faccionalización y al enfrentamiento dentro del propio movimiento feminista. Por tanto, la respuesta feminista debe ser la del cuestionamiento de todo orden vigente, sobre todo si recurre al dualismo sexual hegemónico y cierra el debate a nuevas resignificaciones y perspectivas: «Deconstruir el sujeto del feminismo no es, entonces, censurar su utilización sino, por el contrario, dejar al término libre en un futuro de múltiples significaciones, emanciparlo de las ontologías raciales o maternales a las que ha sido restringido, y darle juego como un sitio donde pueden ver la luz significados aún no previstos. (…)En cierto sentido, lo que las mujeres significan se ha dado por hecho durante demasiado tiempo, y lo que ha sido fijado como el “referente” del término ha sido “fijado”, normalizado, inmovilizado y paralizado en posiciones de subordinación. OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 Feminismo como crítica: sujeto y universalidad (…)Reconfigurar el referente como lo significado, y autorizar o salvaguardar la categoría de las mujeres como un sitio de posibles resignificaciones, es expandir las posibilidades de lo que significa ser mujer, y en este sentido condicionar y posibilitar un sentido de agencia más amplio» (Butler, 2001: 34-35). No es que no haya un fundamento o una referencia, es que el término mujer está en disputa, siempre abierto a la resignificación o a la reformulación, nunca cerrado, como el modelo de democracia radical de Mouffe, en el que los significantes como libertad e igualdad, base del conjunto de valores compartidos por la comunidad política, siempre están abiertos a significaciones y aplicaciones diversas. Se podría decir que el significante “mujer” es, siguiendo la terminología lacaniana que aplican Laclau y Mouffe, un significante flotante, que como otros conceptos políticos en disputa, se abre a interpretaciones diversas y enfrentadas. Pero lo que propone Butler y Mouffe es precisamente difuminar la concreción de este significante para que, apelando al mismo, se esté mencionando a un conjunto de luchas y propuestas políticas que pueden ser muy distintas entre sí pero que comparten un enemigo común, la opresión. En tal caso el significante “mujer” se tornaría el significante OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 210 Carlos Villanueva Castro vacío que aglutina numerosas formas de ser y vivir la sexualidad y la feminidad. Este significante vacío puede ser además el representante de otras muchas luchas que trascienden a las del propio movimiento feminista. Feminismo como crítica: sujeto y universalidad Feminismo como crítica: sujeto y universalidad «Pensemos en el ejemplo clásico de la protesta popular (huelgas, manifestación de masas, boicots) con sus reivindicaciones específicas (“¡No más impuestos!”, “¡Justicia para los encarcelados!”, “¡No más explotación de recursos naturales!”…): la situación se politiza cuando la reivindicación puntual empieza a funcionar como una condensación metafórica de una oposición global contra Ellos, los que mandan, de modo que la protesta pasa de referirse a determinada reivindicación a reflejar la dimensión universal que esa específica reivindicación contiene (de ahí que los manifestantes se suelan sentir engañados cuando los gobernantes, contra los que iba dirigida la protesta, aceptan resolver la reivindicación puntual; es como si, al darles la menor, les estuvieran arrebatando la mayor, el verdadero objetivo de la lucha)» (Zizek, 2010: 43). Pero la universalidad no se constituye a través de procedimientos previos a la acción de carácter universalista, los que proponen Rawls o Habermas, que con una pretensión de neutralidad y un rechazo a las lógicas de poder, como expone Butler en “Fundamentos contingentes” (2001), acaban imponiendo un modelo culturalmente sesgado y realizando un acto de uso de poder: «El término “universalidad” tendría que quedar permanentemente abierto, permanentemente disputado, permanentemente contingente, para no dar por cerrados reclamos futuros de inclusión por adelantado. (…) En este sentido, no estoy acabando con la categoría, sino tratando de aliviar a la categoría de su peso fundamentalista para convertirla en un sitio de disputa política permanente» (Butler, 2001: 18). Esta propuesta, que casa perfectamente con la de Mouffe y Laclau, implica que toda demanda parcial, sea satisfecha o no por el orden dado, puede trascender sus límites y representar un conjunto de demandas con las que, siguiendo la terminología laclauniana, se establezca un lazo equivalencial. Esto para Laclau es la construcción del pueblo que es el sujeto político que configura el populismo, que puede inclinarse en direcciones ideológicas opuestas, pero que siempre se constituye en torno a la confluencia de demandas particulares en torno a una de ellas que se posiciona como el significante vacío, inconcreto y abierto en su significación, que representa al resto de demandas frente a un enemigo común. Esta forma, fundada en la contingencia, de construirse la universalidad, tal como decíamos, supone la superación de los límites de una lucha o demanda concreta, algo que Butler denomina como traducción. FEMINISMO Y UNIVERSALIDAD Lo que Mouffe propone en el artículo que hemos comentado es apostar por el feminismo como una de las muchas luchas sociales que deben confluir en el establecimiento de un nuevo bloque hegemónico que subvierta elementos neoliberales que imperan en el sistema vigente. Algo parecido a lo que junto a Laclau propuso en Hegemonía y estrategia socialista (1985) al denunciar el sustancialismo del marxismo ortodoxo y apostar por una nueva concepción de sujeto político no predefinida en la que caben demandas y propuestas que van más allá del hasta entonces supuesto sujeto de la revolución, la clase obrera. La hegemonía, término adoptado de Antonio Gramsci, supone para ellos dejar de lado el determinismo del materialismo histórico y apostar por influir en el terreno de la superestructura, en el de la cultura y el enfrentamiento ideológico. Es desde ahí desde donde configurar un nuevo eje que articule las distintas demandas: «Without hegemony, socialist practice can focus only on the demands and interests of the working class. But insofar as the dislocation of stages compels the working class to act on a mass terrain, it must abandon its class ghetto and transform itself into the articulator of a multiplicity of antagonisms and demands stretching beyond itself» (Laclau/Mouffe, 1985: 58). Sólo bajo la óptica de la contingencia y rechazando el sustancialismo residual del marxismo clásico es posible apostar por la aglutinación de diversas demandas y luchas en las que, siguiendo el planteamiento de Laclau cuando habla de populismo, una parte que se sabe parte se sitúa en el lugar del universal que nunca podrá ocupar realmente, excepto en el caso de una sociedad totalitaria. Las luchas locales y los movimientos heterogéneos que se dan en diversos lugares del globo pueden poseer entre sí las suficientes semejanzas o compartir un mismo enemigo, el orden neoliberal, o tendente al neoliberalismo, que permite extraer de casos concretos símbolos que representan demandas compartidas. Slavoj Zizek expone en En defensa de la intolerancia (2010), cuestionando las políticas del multiculturalismo, como las luchas parciales poseen en su seno un germen de universalidad que las trasciende: OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 211 Feminismo como crítica: sujeto y universalidad Laclau insiste en la semejanza de ese concepto con su propuesta del lazo equivalencial cuando aún no había articulado una teoría OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 212 Carlos Villanueva Castro elaborada sobre el populismo pero ya defendía la necesidad de la izquierda por articular un bloque contra-hegemónico en el ámbito de los discursos y las prácticas: elaborada sobre el populismo pero ya defendía la necesidad de la izquierda por articular un bloque contra-hegemónico en el ámbito de los discursos y las prácticas: «Now, I think that the internal structure of what Butler calls “translation” and what i call “equivalence” is very close indeed. Translation, for her, means the deterritorialization of a certain content by adding something which, being outside the original context of enunciation, universalizes itself by multiplying the positions of enunciation from which that content derives its meaning. A feminist discourse claiming women´s rights in the name of human equality does exactly that. (…) Well, a relation of equivalence, in the sense that I understand it, performs exactly that role» (Laclau, 2000: 194). Del mismo modo que dentro del concepto de mujer caben múltiples significados que siempre estarán en disputa, y que el movimiento feminista aglutina perspectivas muy diversas pero que comparten una meta común, el feminismo puede no ser concebido como una lucha parcial y concreta y establecerse como un representante más, junto a otras luchas, del esfuerzo por un nuevo orden social, siempre que comparta con ellas el rechazo a la opresión y a la desigualdad. Mouffe en su artículo apuesta por la vinculación de los movimientos en defensa de las mujeres, los trabajadores, las minorías étnicas, las minorías sexuales, el ecologismo y demás luchas sociales en un “nosotros” que, sin borrar las diferencias, constituya una comunión conveniente en la lucha por un nuevo orden hegemónico, la lucha por tratar de convencer sobre nuevas formas de entenderse y vivir, así como de por establecer políticas y legislaciones favorables a estas demandas. Esta lucha, que puede plantearse tanto dentro como fuera de las instituciones, aunque Mouffe piensa generalmente en la importancia de trabajar por el cambio dentro de las instituciones, necesita del movimiento feminista para establecer sus pretensiones y configurar esa nueva universalidad abierta y contingente de la que venimos hablando. OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 Feminismo como crítica: sujeto y universalidad En la construcción de una nueva hegemonía el feminismo, por su larga historia de lucha contra la opresión, debe entenderse como un tipo de pensamiento y praxis que va mucho más allá de la mera consecución de objetivos parciales de un determinado colectivo, que no es otro que la mitad de la población mundial, sino como un pilar, entre otros tantos, que sustentan el proyecto de un mundo más justo y una lucha que ejemplifica y reúne a todas las demás. OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 213 Feminismo como crítica: sujeto y universalidad ZIZEK, Slavoj (2010). En defensa de la intolerancia, Madrid: Diario Público. OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 BIBLIOGRAFÍA BUTLER, Judith (1998). “Actos performativos y constitución de género: un ensayo sobre fenomenología y teoría feminista”. Debate Feminista, 17, pp. 296-314. -(2001). “Fundamentos contingentes”. La Ventana, 13 (2), pp. 7-41. -(2007). El género en disputa, Madrid: Paidós. LACLAU, Ernesto Y MOUFFE, Chantal (1985). Hegemony and Socialist strategy. Towards a radical democratic politics. Londres: Verso. LACLAU, Ernesto (2005) La razón populista. Buenos Aires: Fondo de Cultura Económica. -(2000) “Structure, History and the Political” en BUTLER, Judith LACLAU, Ernesto y ZIZEK, Slavoj (2000). Contingency, Hegemony, Universality. Contemporary debates on the Left, Londres: Verso. MOUFFE, Chantal (1996). “Feminismo, ciudadanía y política democrática radical” en BELTRÁN, Elena y SÁNCHEZ, Cristina (eds) (1996), Las Ciudadanas y lo político, Instituto Universitario de Estudios de la Mujer, Madrid: UAM. ZIZEK, Slavoj (2010). En defensa de la intolerancia, Madrid: Diario Público. OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213 OXÍMORA REVISTA INTERNACIONAL DE ÉTICA Y POLÍTICA NÚM. 7. OTOÑO 2015. ISSN 2014-7708. PP. 202-213
https://openalex.org/W2758910468	https://europepmc.org/articles/pmc5634655?pdf=render	English	null	Estimating the burden of scrub typhus: A systematic review	PLoS neglected tropical diseases	2,017	cc-by	9,990	RESEARCH ARTICLE Ana Bonell1, Yoel Lubell2,3, Paul N. Newton3,4, John A. Crump5, Daniel H. Paris2,3,6,7* Ana Bonell1, Yoel Lubell2,3, Paul N. Newton3,4, John A. Crump5, Daniel H. Paris2,3,6,7* 1 Oxford University Clinical Research Unit, National Hospital of Tropical Diseases, Hanoi, Vietnam, 2 Mahidol Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand, 3 Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom, 4 Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit, Microbiology Laboratory, Mahosot Hospital, Vientiane, Laos, 5 Centre for International Health, University of Otago, Dunedin, New Zealand, 6 Department of Medicine, Swiss Tropical and Public Health Institute, Basel, Switzerland, 7 Faculty of Medicine, University Basel, Basel, Switzerland a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Methods Received: March 13, 2017 Accepted: July 28, 2017 Published: September 25, 2017 We systematically reviewed all literature from public health records, fever studies and reports available on the Ovid MEDLINE, Embase Classic + Embase and EconLit databases, to estimate the burden of scrub typhus since the year 2000. Copyright: © 2017 Bonell 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. Findings In prospective fever studies from Asia, scrub typhus is a leading cause of treatable non- malarial febrile illness. Sero-epidemiological data also suggest that Orientia tsutsugamushi infection is common across Asia, with seroprevalence ranging from 9.3%–27.9% (median 22.2% IQR 18.6–25.7). A substantial apparent rise in minimum disease incidence (median 4.6/100,000/10 years, highest in China with 11.2/100,000/10 years) was reported through passive national surveillance systems in South Korea, Japan, China, and Thailand. Case fatality risks from areas of reduced drug-susceptibility are reported at 12.2% and 13.6% for South India and northern Thailand, respectively. Mortality reports vary widely around a median mortality of 6.0% for untreated and 1.4% for treated scrub typhus. Limited evidence suggests high mortality in complicated scrub typhus with CNS involvement (13.6% mortal- ity), multi-organ dysfunction (24.1%) and high pregnancy miscarriage rates with poor neona- tal outcomes. * daniel.paris@swisstph.ch Background Scrub typhus is a vector-borne zoonotic disease that can be life-threatening. There are no licensed vaccines, or vector control efforts in place. Despite increasing awareness in endemic regions, the public health burden and global distribution of scrub typhus remains poorly known. Citation: Bonell A, Lubell Y, Newton PN, Crump JA, Paris DH (2017) Estimating the burden of scrub typhus: A systematic review. PLoS Negl Trop Dis 11(9): e0005838. https://doi.org/10.1371/journal. pntd.0005838 Scrub typhus disease burden Interpretation Competing interests: The authors have declared that no competing interests exist. Scrub typhus appears to be a truly neglected tropical disease mainly affecting rural popula- tions, but increasingly also metropolitan areas. Rising minimum incidence rates have been reported over the past 8–10 years from countries with an established surveillance system. A wider distribution of scrub typhus beyond Asia is likely, based on reports from South Amer- ica and Africa. Unfortunately, the quality and quantity of the available data on scrub typhus epidemiology is currently too limited for any economical, mathematical modeling or mapping approaches. Author summary Scrub typhus is a mite-transmitted infectious disease that can be life-threatening. Diag- nosing this disease is difficult, requiring special techniques that are often not readily available. As the actual impact of scrub typhus on the population and its geographical dis- tribution remains unknown, we searched systematically for available information in medi- cal databases. Scrub typhus is common: more than every fifth person in areas where scrub typhus occurs carry antibodies as a sign of previous contact. All countries with an estab- lished surveillance system have recorded an increase in scrub typhus cases over the past 8–10 years, while reports from South America and Africa suggest a wider distribution beyond Asia. Scrub typhus is a serious disease: approximately 6% of cases die if untreated, and 1.5% if treated, but mortality can reach 13% in areas where the usual treatment does not always work well. Death rates of complications are higher, reaching 14% in brain infections, 24% with multiple organ failure, and pregnancies with scrub typhus can have poor outcomes, with high miscarriage rates. Despite many limitations on the amount and quality of available reports, we found that scrub typhus is a severely underappreciated tropical disease, affecting mainly rural populations, but increasingly urban areas as well. Data Availability Statement: All relevant data are within the paper and its Supporting Information file. Data Availability Statement: All relevant data are within the paper and its Supporting Information file. Funding: Mahidol Oxford Tropical Medicine Research Unit (MORU), Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit (LOMWRU) and Oxford University Clinical Research Unit Vietnam (OUCRU) are in part supported by the Wellcome Trust, UK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1 / 17 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0005838 September 25, 2017 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0005838 September 25, 2017 Scrub typhus disease burden Although scrub typhus received much attention before and during the Second World War and to a lesser degree during the Vietnam/American war, basic epidemiology is poorly under- stood with limited data on incidence and burden of disease for patients, their families, societies and the economy. This ignorance is probably due to a combination of factors; clinical presen- tation is very similar to other causes of fever, diagnostic difficulties contribute to mis-diagnosis and under recognition, and appropriate diagnostic tests are not widely available. Following the discovery of chloramphenicol in the 1940s, the scientific interest dropped rapidly and scrub typhus has since received little global attention [12]. The data quoted by the World Health Organization (WHO) stating that over a billion people are at risk and one million cases are estimated per year is referenced to a paper published 20 years ago in 1997 [13, 14]. Extrapolation based on geographical mite distributions and densities are not helpful due to patchy data, limited by the dynamics of infected mite populations and insufficient characteri- zation of transmitting vectors. With new data and improvements in approaches to estimating the burden of febrile illnesses, it is important to reevaluate the burden of scrub typhus. Rationale for this study: Scrub typhus is among the leading causes of undifferentiated treat- able fever in Asia. The mortality rates appear low at first glance, but considering the numbers of those exposed and/or infected a significant disease burden is expected globally. The follow- ing research questions were addressed: What is the estimated global burden of disease for scrub typhus? What data on seroprevalence and minimum incidence for scrub typhus are available by geographical regions? What data on DALYs, YLLs and YLDs are available, and what is the mortality rate of treated scrub typhus? In this study we summarized the literature relating to the disease burden and economic impact of scrub typhus since the year 2000 in order to estimate the global incidence and bur- den of this disease. Introduction Scrub typhus is an infectious disease caused by Orientia tsutsugamushi, an obligate intracellular bacteria, transmitted by the bites of chigger mites [1]. In Southeast Asia, scrub typhus is a lead- ing cause of treatable non-malarial febrile illness [2]. The first accounts linking febrile illness with the appearance of “harmful” mites (Japanese: “tsutsuga” mushi) range back to 313 AD in China [3]. Scrub typhus was originally associated with the Asian-Pacific “Tsutsugamushi trian- gle,” until recent evidence from the Arabian Peninsula, Chile and possibly Kenya suggested a wider global distribution in tropical and subtropical regions [4–7]. The use of improved diagnostic methods, increased medical investigations and awareness have recently contributed to greater recognition of scrub typhus in some countries, such as in Laos, India, southern China, South Korea, and Japan [8]. There is also evidence suggesting that a combination of climate change and expansion of humans into previously uninhabited areas may play a role in both re-emergence and apparent rising incidence of scrub typhus [9– 11]. There are no licensed vaccines for scrub typhus, and no systematic vector control efforts in place. Despite increasing awareness in endemic regions, the public health burden and global distribution of scrub typhus remains poorly known. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0005838 September 25, 2017 2 / 17 Papers were also graded on diagnostic tests used: Papers were also graded on diagnostic tests used: 1. Pathogen detection through cell culture/animal inoculation. PCR positivity. IFA serological diagnosis with either sero-conversion or fourfold antibody response. 2. Single high Weil Felix titre of 1:100 for all samples / Single high IFA titre >1:50 for all samples / IgM or IgG ELISA positive. 3. Single Weil Felix or IFA titre, non-significant for all patients / No record of significant Weil Felix or IFA titre / Clinical diagnosis / unspecified. Methods A literature search of three databases: Ovid MEDLINE (2000-present), Embase Classic + Embase (2000-present) and EconLit (2000-present) was conducted on 11th April 2016 using three search strategies. First search terms: Scrub typhus, Orientia tsutsugamushi, Rickettsia tsu- tsugamushi, chigger borne rickettsiosis, chigger borne typhus, Orientia tsutsugamushi infec- tion, Rickettsia tsutsugamushi infection, tsutsugamushi disease, tsutsugamushi fever (keyword) AND prevalence, incidence, epidemiology. A second search included the above search for scrub typhus and all variations AND cost, cost analysis, cost of illness, drug costs, economics, health care cost, hospital costs, cost benefit analysis, cost effectiveness analysis, quality adjusted life year. A third search included scrub typhus AND mortality or death on the 1st Oct 2016, for which all currently available data was included (no date restrictions). Data on untreated mor- tality have been reported [15], and therefore only papers with treated infection were included in estimating mortality. All titles and abstracts were reviewed by 2 authors for inclusion and any disagreements were discussed and inclusion based on the senior author’s opinion. Only English language publications were included. A total of 190 publications were selected for full article review (Fig 1A and 1B, S1 File). The final number of articles included for full data extraction was 87. The data extraction form was trialed on the first 5 papers and required minor alterations. Due to the limited nature of data available no summary measures were applied. Studies were examined for selection bias and graded as follows: 1. Population based active surveillance / community surveillance / hospital surveillance. 1. Population based active surveillance / community surveillance / hospital surveillance. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0005838 September 25, 2017 3 / 17 Scrub typhus disease burden Fig 1. PRISM flow diagrams summarizing the search results. Panel A, search for scrub typhus epidemiology and health economics from 2000 onwards. Panel B, search for scrub typhus mortality with no date restrictions. Flow diagrams were downloaded from www.prisma-statement.org. https://doi.org/10.1371/journal.pntd.0005838.g001 Fig 1. PRISM flow diagrams summarizing the search results. Panel A, search for scrub typhus epidemiology and health economics from 2000 onwards. Panel B, search for scrub typhus mortality with no date restrictions. Flow diagrams were downloaded from www.prisma-statement.org. https://doi.org/10.1371/journal.pntd.0005838.g001 https://doi.org/10.1371/journal.pntd.0005838.g001 https://doi.org/10.1371/journal.pntd.0005838.g001 2. Prospective, consecutive patient case series with no inappropriate exclusions / retrospective case series / non-consecutive case series / reference laboratory series. 3. Exclusion of patients (i.e. most unwell patients treated) likely to significantly affect outcome / summary of case reports from literature. Papers were also graded on diagnostic tests used: Incidence surveillance data Five countries report a passive national surveillance system for scrub typhus. In South Korea scrub typhus was designated a group III notifiable disease (requiring man- datory reporting and routine monitoring) in 1994. Cases are confirmed by the Korean Centre for Disease Control and Prevention (KCDC) and must show one of the following: an increase in the IFA IgM to O. tsutsugamushi of 1:16; an increase in the anti-O. tsutsugamushi IFA IgG titre to 1:256; a 4 fold increase in IFA titre. Data from KCDC suggest that the annual minimum incidence increased from 5.7 to 17.7/100,000 people from 2001 to 2012 (>3-fold) (Table 1) [16–18]. Interestingly, the number of patients recorded in urban areas has also increased dramatically, for example, the annual minimum incidence in Ulsan Metropolitan City increased from 2.8/100,000 in 2003 to 59.7/100,000 in 2013 (>21 fold). In Seoul there is evidence of urban scrub typhus, further demonstrating the changing geographical scope and habitat of infected chigger mites [16]. In Japan scrub typhus is a notifiable disease and must be reported to the National Epidemi- ological Surveillance of Infectious Diseases (NESID) within 7 days of diagnosis by a physician. Confirmed cases are based on: isolation or identification of the organism in the blood; PCR positivity; detection of serum IgM; a 4 fold increase in IFA titre. Data from NESID show an increase of annual minimum incidence from 0.6/100,000 in 2000 to 3.6/100,000 in 2008 (6-fold) [19, 20]. In Thailand, scrub typhus patients have been reported to the Bureau of Epidemiology for the last 30 years. Data can be viewed online on the homepage available under URL: http:// www.boe.moph.go.th/boedb/surdata/disease.php?dcontent=situation&ds=44 Cases are defined based on one or more of the following: isolation or identification of the organism in the blood or tissue sample; PCR positivity; a 4 fold increase in IFA titre (IgG and/or IgM); 1:400 IFA in acute serum (IgG and/or IgM); IgM ELISA positivity. Data from the Bureau of Epidemiology noted an increase of annual minimum incidence from 6.0/ 100,000 in 2003 to 17.1/100,000 in 2013 (2.9 fold) [21]. In China, scrub typhus is a notifiable disease that must be reported to the China Center for Disease Control and Prevention. Scrub typhus disease burden Incidence surveillance data Cases are defined as those with clinically compatible infection and one or more of the following; isolation or identification of the organism in a blood or tis- sue sample; PCR positivity; a 1:160 Weil-Felix test; a 4 fold increase in IFA titre (IgG and/ or IgM). The reported countrywide minimum incidence increased from 0.1/100,000 to 1.1/ 100,000 people/year from 2006 to 2014 (>11-fold) [22]. The reported incidence rates vary widely by region with the southern provinces more affected. Guangdong Province saw an increase in reported annual minimum incidence from 0.4/100,000 to 3.6/100,000 people from 2006 to 2013 (>8-fold), whereas in 2012 the provinces of Laiwu and Guangzhou City had annual incidences of 5.5/100,000 and 9.9/100,000 people, respectively [10, 23–25]. Seroprevalence data There are seroprevalence data available from Bangladesh, Indonesia, Laos, Malaysia, Papua New Guinea and Sri Lanka (Table 1). Seropositivity ranged from 9.3%–27.9% suggesting high background exposure levels to O. tsutsugamushi in these countries [26–31] Results Of the 87 studies included, 44 (50.6%) gave information on incidence, seroprevalence and/or prevalence in febrile inpatients (denominator = febrile cases per year), whilst health economic or burden of disease data were given in 4 studies (4.6%) and mortality data in 38 (43.7%). The publications with no apparent patient selection bias and use of grade A evidence to diagnose scrub typhus were few (16/87, 18.4%). A total of 143,544 patients with scrub typhus were described in the included papers. Females were reported to be more commonly infected than males 77,204 versus 57,535 (57.3% versus 42.7%, respectively). PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0005838 September 25, 2017 4 / 17 https://doi.org/10.1371/journal.pntd.0005838.t001 Kenya were subject to less bias as they included complete prospective series of patients present- ing with fever to healthcare facilities and demonstrated rates from 1.8–22.3% (Table 2). Kenya were subject to less bias as they included complete prospective series of patients present- ing with fever to healthcare facilities and demonstrated rates from 1.8–22.3% (Table 2). Data from specific sub-populations are presented in Table 3. Two studies describe the importance of scrub typhus in women during pregnancy from Laos and the Thai-Myanmar border—with scrub typhus occurring in 3.6–5.4% of febrile patients [32–34]. Maternal infec- tion with scrub typhus during pregnancy was associated with poor maternal and fetal out- comes; 2/9 (22.2%) of cases in Laos and 4/11 (36.4%) in Thailand/Myanmar suffered either abortion or stillbirth. Among Lao patients with meningitis/encephalitis, 16.0% of those with a diagnosed bacterial cause for their infection had evidence for scrub typhus [35]. However, only 54.8% of these patients received treatment with appropriate antimicrobials during admission and the mortal- ity rate associated with CNS complications was 13.6%. There are no data on morbidity or long-term sequelae available. Scrub typhus disease burden Table 1. Estimates of incidence and sero-prevalence per country. First Author Country Incidence/ 100,000 Year data collected Total number infected Patient Selection Grade Diagnostic Grade Park South Korea 17.7 2012 8,604 1 A Yasunaga Japan 3.6 2007–2008 210 3 A Wu China 1.22 2014 16,050 1 A Lee Taiwan 14.3 2000–2004 1,396 1 A N/A Thailand 11.8 2015 7,696 1 A First Author Country, Region Sero- prevalence Year data collected Total number tested Patient Selection Grade Seroprevalence Diagnostic Grade Maude Bangladesh 23.7% 2010 1,209 1 B Richards Indonesia, Gag Island 9.3% 2003 53 2 B Valle´e Laos, Vientiane 20.7% 2006 2,002 1 B Tay Malaysia, Western Malaysia 17.9% 2007–2010 280 2 B Spicer Papua New Guinea 27.9% 2001 140 2 B Premaratna Sri Lanka 26.3% 2008 57 3 B Patient selection grade: Grade I = Population based active surveillance / community surveillance / hospital surveillance, Grade II = Prospective, consecutive patient case series with no inappropriate exclusions / non-consecutive case series / retrospective case series / reference laboratory series, Grade III = Exclusion of patients (i.e. most unwell patients treated) likely to significantly affect outcome / summary of case reports from literature.Diagnostic grade: Grade A = Pathogen detection through cell culture/animal inoculation. PCR positivity. IFA serological diagnosis with either sero-conversion or fourfold antibody response, Grade B = single sample with high titre/ELISA IgG/IgM, Grade C = Single high Weil Felix or IFA titre but titre non-significant for all patients / No record of significant Weil Felix or IFA titre / clinical diagnosis / unspecified. Table 1. Estimates of incidence and sero-prevalence per country. timates of incidence and sero-prevalence per country Patient selection grade: Grade I = Population based active surveillance / community surveillance / hospital surveillance, Grade II = Prospective, consecutive patient case series with no inappropriate exclusions / non-consecutive case series / retrospective case series / reference laboratory series, Grade III = Exclusion of patients (i.e. most unwell patients treated) likely to significantly affect outcome / summary of case reports from literature.Diagnostic grade: Grade A = Pathogen detection through cell culture/animal inoculation. PCR positivity. IFA serological diagnosis with either sero-conversion or fourfold antibody response, Grade B = single sample with high titre/ELISA IgG/IgM, Grade C = Single high Weil Felix or IFA titre but titre non-significant for all patients / No record of significant Weil Felix or IFA titre / clinical diagnosis / unspecified. Fever studies There are several case series describing the frequency of scrub typhus among patients present- ing with fever. In India, scrub typhus was the causative agent in 16.1–96.9% of febrile patients presenting to hospitals (Table 2). However, these studies all suffer from selection bias, as other causes of febrile illness had already been excluded. Studies from Cambodia, Laos, Nepal, and PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0005838 September 25, 2017 5 / 17 Patient selection grade: Grade I = Population based active surveillance / community surveillance / hospital surveillance, Grade II = Prospective, consecutive patient case series with no inappropriate exclusions / non-consecutive case series / retrospective case series / reference laboratory series, Grade III = Exclusion of patients (i.e. most unwell patients treated) likely to significantly affect outcome / summary of case reports from literature.Diagnostic grade: Grade A = Pathogen detection through cell culture/animal inoculation. PCR positivity. IFA serological diagnosis with either sero-conversion or fourfold antibody response, Grade B = single sample with high titre/ELISA IgG/IgM, Grade C = Single high Weil Felix or IFA titre but titre non-significant for all patients / No record of significant Weil Felix or IFA titre / clinical diagnosis / unspecified. Scrub typhus disease burden Table 2. Data on epidemiology of scrub typhus from fever studies (hospital based incidence). First Author Country, region Year Patients with scrub typhus n (%) Total patients in study Patient selection grade Diagnostic Grade Roopa [40] India, Pondicherry 2012– 2015 225 (41.3%) 545 2 B Borkakoty [41] India, Arunachal Pradesh 2013 31 (96.9%) 32 3 B Kumar [38] India, Chandigarh 2011– 2012 49 (24.4%) 201 2 A Narvenkar [42] India, Goa 2009– 2010 15 (34.1%) 44 2 B Chrispal [43, 44] India, Vellore 2007– 2008 189 (47.5%) 398 2 B Kamarasu [45] India, Tamil Nadu 2004– 2005 204 (16.1%) 1,270 2 B Sharma [46] India, Himachal Pradesh 2003– 2004 52 (34.7%) 150 2 B Vaz [47] India, Jammu 2002 12 (50.0%) 24 2 B Mueller [48] Cambodia, remote Western and Eastern provinces 2008– 2010 54 (3.7%) 1,475 2 A Kasper [49] Cambodia, within 50km Phnom Phenh 2006– 2009 35 (1.8%) 1,906 2 A Mayxay [50] Laos, northwest Laos and southern Laos 2008– 2010 122 (6.5%) 1,938 2 A Blacksell [51] Nepal, Kathmandu 2002– 2004 23 (22.3%) 103 2 A Reller [52] Sri Lanka, southern 2007 9 (1.0%) 883 2 A Susilawati [53] Australia, Cairns 2008– 2011 2 (0.6%) 340 2 A Thiga [54] Kenya, 6 regions 2015 76 (4.8%) 1,401 2 B Table 2. Data on epidemiology of scrub typhus from fever studies (hospital based incidence). III = Exclusion of patients (i.e. most unwell patients treated) likely to significantly affect outcome / Summary of case reports from literature. Diagnostic grade: Grade A = Pathogen detection through cell culture/animal inoculation. PCR positivity. IFA serological diagnosis with either sero-conversion or fourfold antibody response, Grade B = single sample with high titre/ELISA IgG/IgM, Grade C = Single high Weil Felix or IFA titre but titre non-significant for all patients / No record of significant Weil Felix or IFA titre / clinical diagnosis / unspecified. Table 3. Publications investigating scrub typhus affected specific sub-populations. First Author Country Year Population specifics Number ST (%) Diagnostic Grade Dittrich et al. [35] Laos, Vientiane 2003– 2011 Patients enrolled if admitted with CNS infections and lumbar puncture indicated 31/1051 (2.9%) A Chansamouth et al. [34] Laos, Vientiane, 2006– 2010 All febrile pregnant inpatients 9/250 (3.6%) A McGready et al. [32] Thai-Burmese border 2004– 2006 All febrile pregnant patients 11/203 (5.4%) A Premanatna et al. At risk population National surveillance data from patients in China, Japan, Korea and Taiwan suggest that the age group of 60–69 years was at highest risk of scrub typhus [18, 20, 22, 36]. In Thailand those aged 45–54 years were most commonly infected. In Japan and Thailand males were more at risk of scrub typhus but in all other countries with reports, females are more at risk. In South Korea, China, Taiwan and Thailand farmers were most at risk (38,183/54,558–70% of infec- tions in China from 2006–2014); unfortunately such data are lacking from Japan. Age stratifi- cation in untreated mortality revealed increasing risk with increasing age, with the age classes 51–60 and >60 years old associated with a 45.6% and 59.8% mortality rate respectively [15]. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0005838 September 25, 2017 6 / 17 Patient selection grade: Grade I = Population based active surveillance / community surveillance / hospital surveillance, Grade II = Prospective, consecutive patient case series with no inappropriate exclusions / non-consecutive case series / retrospective case series / reference laboratory series, Grade III = Exclusion of patients (i.e. most unwell patients treated) likely to significantly affect outcome / Summary of case reports from literature. Diagnostic grade: Grade A = Pathogen detection through cell culture/animal inoculation. PCR positivity. IFA serological diagnosis with either sero-conversion or fourfold antibody response, Grade B = single sample with high titre/ELISA IgG/IgM, Grade C = Single high Weil Felix or IFA titre but titre non-significant for all patients / No record of significant Weil Felix or IFA titre / clinical diagnosis / unspecified. Case fatality ratios Case fatality ratios vary widely between countries, with those countries with easily accessible and established health systems showing lower mortality rates compared to countries with lim- ited facilities (Fig 2 and Table 4). In a previous review, untreated scrub typhus infection was associated with an estimated mortality of 6.0% (median, range 0–70.0%) [15]. This review of treated scrub typhus, which included 39 studies and 91,692 patients found a median mortality of 1.4% (range 0–33.3%). Scrub typhus disease burden Complications and sequelae The long-term impact of infection with scrub typhus has barely been examined. In Taiwan the hazard ratio of developing acute coronary syndrome was 1.4 (95% CI 1.1–1.8) in those with previous infection with scrub typhus compared to the general population without [37]. A recent case series from India that included patients with unexplained fever and/or multi-sys- tem involvement, found 24.4% to have scrub typhus, and 53.1% of patients with scrub typhus had acute kidney injury [38]. A retrospective cohort of severe scrub typhus cases admitted to an ICU in South India, found that respiratory complications requiring mechanical ventilation occurred in 87.9%, and that dysfunction of 3 or more organ systems occurred in 85.2% [39]. [29] Sri Lanka 2008 Consecutive admission of military personnel with fever 26/49 (53%) B Nadjm et al. [55] Vietnam, northern Vietnam 2001– 2003 Consecutive patients admitted to national referral hospital of infectious diseases, with no immediate diagnosis 251/7226 (3.5%) B https://doi.org/10.1371/journal.pntd.0005838.t003 Table 3. Publications investigating scrub typhus affected specific sub-populations. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0005838 September 25, 2017 7 / 17 Disability Adjusted Life Year (DALY) data The burden of disease data for scrub typhus is highly limited. Only one study, from Laiwu Province in China, has calculated the DALYs associated with scrub typhus [24]. This study estimated that 13 DALYs were lost due to scrub typhus across the province (6 in males, 7 in females at a rate of 1.06/100,000). However, in this province no deaths were reported and Fig 2. Case fatality (%) of scrub typhus reported in published case series and studies. This figure shows the locations from included case series and study reports in a map; the case fatality (%) reported is color coded (blue 0–1%; pale blue 1.2–6.3%; pale red 6.6–13.8%; and red 14–33.3%), and the detailed data of the studies included is summarized in Table 4. Source of map: http://www.naturalearthdata.com, accessed on the 14th July 2017. Kashmir and Arunachal Pradesh are depicted in grey with borders as red dashes, due to either disputed or indeterminate areas. h //d i /10 13 1/j l d 000 838 002 Fig 2. Case fatality (%) of scrub typhus reported in published case series and studies. This figure shows the locations from included case series and study reports in a map; the case fatality (%) reported is color coded (blue 0–1%; pale blue 1.2–6.3%; pale red 6.6–13.8%; and red 14–33.3%), and the detailed data of the studies included is summarized in Table 4. Source of map: http://www.naturalearthdata.com, accessed on the 14th July 2017. Kashmir and Arunachal Pradesh are depicted in grey with borders as red dashes, due to either disputed or indeterminate areas. https://doi.org/10.1371/journal.pntd.0005838.g002 https://doi.org/10.1371/journal.pntd.0005838.g002 8 / 17 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0005838 September 25, 2017 Scrub typhus disease burden Table 4. Overview of case fatality ratios reported for treated scrub typhus. Date of study, Author Country No. with ST CFR % Comments 2006–2012, Yang China 308 0 DALY study in Laiwu 1995–2006, Liu China 480 0 Fever study 2006–2014, Wu China 54,558 0.07 National surveillance data 2012, Wei China 29 13.8 Outbreak investigation in a Guangzhou city park 2002, Vaz India 12 0 Outbreak investigation in soldiers 2014, Luthra India 197 1.0 Fever study 2011, Gurung India 63 1.6 Fever study 2008–2012, Thomas India 262 1.9 Fever study in children 2010–2011, Mahajan India 253 5.1 Fever study 2013, Masand India 30 6.6 Fever study 2009–2010, Varghese India 154 7.8 Fever study 2005–2010, Varghese India 623 9.0 Fever study 2014, Khandelwal India 52 9.6 Fever study in children 2007–2008, Chrispal India 189 12.2 Fever study 2013–14, Sharma India 228 13.6 Fever study 2011–2012, Kumar India 49 16.3 Fever study 2014, Singh India 13 23.1 ICU patients with pulmonary complications 2003–2004, Sharma India 52 28.8 Outbreak investigation Himachal Pradesh 2009–2010, Narvencar India 15 33.3 Fever study 2000, Matsui Japan 756 0.3 National surveillance data 2007–2008, Yasunaga Japan 210 1.0 Retrospective use of discharge data 2003–2012, Yoo South Korea 141 0 Hospital surveillance data 1985–1990, Yi South Korea 189 0 Clinical diagnosis only 2008–2012, Park South Korea 30,478 0.1 National surveillance data 2004–2006, Kim South Korea 208 0.5 Case control study at single site 2001–2011, Jang South Korea 771 1.4 Retrospective study in single site 2000–2006, Lee South Korea 297 6.1 Retrospective study in single site 2000–2006, Kim South Korea 160 6.3 Scrub typhus in patients with liver disease 2003–2011, Dittrich Laos 31 13.6 Patients with CNS symptoms 1948–1950, Bailey Malaysia 35 0 Early trials of antibiotic use 2008, Premaratna Sri Lanka 26 0 Outbreak investigation in soldiers 2006–2010, Wang Taiwan 126 0 Retrospective study in single site 1952, Prezyman Taiwan 47 0 Early trial of antibiotic use 1996, Watt Thailand 19 0 Prospective evaluation of antimicrobial response 1985–2002, Silpapojakul Thailand 73 1.4 Case series of children 2001–2002, Thap Thailand 18 16.7 Patients with septic shock 2011–2012, Sriwongpan Thailand 257 13.6 Retrospective hospital series 1965, Hazlett Vietnam 32 0 Case series from military hospital 2001–2003, Nadjm Vietnam 251 1.2 Hospital surveillance data htt //d i /10 1371/j l td 0005838 t004 Table 4. Overview of case fatality ratios reported for treated scrub typhus. https://doi.org/10.1371/journal.pntd.0005838.t004 Scrub typhus disease burden Discussion Scrub typhus represents a major cause of treatable febrile illness across Asia, but its disease incidence remains elusive. Fever remains one of the major reasons to seek healthcare in tropi- cal regions but their causes remain ill-defined [57]. Access to updated evidence on incidence and trends for common causes of febrile illnesses is essential for guiding and informing global, regional, and national health policies. This systematic review collated all currently available lit- erature regarding the disease burden and economic impact of scrub typhus and the result is sobering; there are very few studies and they have great heterogeneity in methodology. Acquisition of estimates for incidence and mortality proved difficult, as numerators had varying levels of confidence in diagnosis or denominators were either absent, or required fur- ther extrapolation. Ideally, data derived from population-based surveillance studies would be graded considerably higher than from hospital-based surveillance, but unfortunately no non- hospital-based surveillance data are publicly available for scrub typhus—unlike for diseases like typhoid where these data are readily available for various countries [58]. Further, the epi- demiology of scrub typhus within a country is heterogeneous–the pronounced seasonality of these diseases and the changing urban/rural distribution, with defined areas of high infected mite intensities (mite islands) challenge the common approaches of disease incidence evalua- tion [59, 60]. Febrile illness surveillance should be performed in multiple representative areas, ideally covering one full calendar year before inferences on national disease incidence can be made [58]. Incidence surveillance Only 5 countries have established scrub typhus surveillance systems. All of these have shown an increasing minimum incidence of scrub typhus over recent years, with increasing evidence of shift towards urbanized areas. However, the apparent increase in minimum incidence is confounded by local enhanced knowledge of the disease and it remains uncertain whether these data reflect true de novo emerging disease or emerging awareness of a pre-existing dis- ease. Surveillance systems also use diverse diagnostic tests and therefore inter-country compar- isons are not always possible. There are no data on whether these surveillance systems have been evaluated to determine an estimate of missed cases, however it is likely that the numbers are conservative estimates. Regardless of these flaws, surveillance systems are an essential part of disease control strategies. Improved febrile disease surveillance providing national data should be initiated in more afflicted countries, as this would result in morbidity and mortality data that could be used to direct healthcare resources, future vaccine demand and delivery and assessment of effectiveness of any control programs. Clearly, striving towards improved surveillance should be key, with a focus on providing reliable numerators (using diagnostic assays with suitable sensitivities and specificities), and representative denominators (well-defined target populations). Additionally, no ‘multiplier data’ or ‘multiplier studies’ are available—these are considered to improve estimation of inci- dence by using healthcare utilization surveys and to correct for under-ascertainment in health- care facility studies [58]. therefore these data cannot be extrapolated to countries such as India or Laos with evidence of scrub typhus associated mortality. A South Korean study evaluating the net benefit of a scrub typhus prevention program, estimated the cost of scrub typhus (medication and hospital costs and loss of earnings) at $6.6 million per year in 2008 [56]. However, scrub typhus mortality in South Korea was only 0.14% and 75% of patients with a diagnosis were hospitalized. Therefore, these figures cannot be applied to other economically poorer countries where health practice is very different [17]. therefore these data cannot be extrapolated to countries such as India or Laos with evidence of scrub typhus associated mortality. A South Korean study evaluating the net benefit of a scrub typhus prevention program, estimated the cost of scrub typhus (medication and hospital costs and loss of earnings) at $6.6 million per year in 2008 [56]. However, scrub typhus mortality in South Korea was only 0.14% and 75% of patients with a diagnosis were hospitalized. Therefore, these figures cannot be applied to other economically poorer countries where health practice is very different [17]. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0005838 September 25, 2017 9 / 17 Fever studies Scrub typhus is a leading cause of treatable non-malarial febrile illness in prospective fever eti- ology studies (n = 14). An increasing number of studies have unraveled the major contribution of scrub typhus to the febrile illness burden. However, the large variation of scrub typhus rates in prospective fever studies (median 23.4% IQR 5.2–39.7 ranging from 1–96.9% depending on country and patient selection), reflect a lack of standardization and comparability among study designs and diagnostic modalities used. None of these studies have used modeling or extrapolation to take into account data from healthcare utilization surveys, which may give a more accurate idea of numbers of people with scrub typhus. In addition, recent studies have raised concern on the persistence of O. tsutsugamushi after treatment, especially using bacteri- ostatic drugs such as tetracyclines and macrolides [63, 64]. Health economics DALY data are lacking in all countries except from one area of China, where a rate of 1.06/ 100,000 people was found, with a zero mortality rate. Case series and studies from Taiwan and India examining long-term complications, imply that the mortality and morbidity from scrub typhus is under-recognized and that possible long term consequences may occur many years later, and may be important contributors to the overall DALY burden [37, 67]. Despite scrub typhus being the foremost cause of treatable febrile illness in Asia it is not evaluated by the Global Burden of Disease studies [68]. Vulnerable populations and case fatality ratios Based on very limited data, scrub typhus is likely to have considerable impact on vulnerable populations–the median untreated mortality of scrub typhus in the elderly was ~29%—approxi- mately 5-fold higher compared to the overall population mortality of 6% [15]. In women with scrub typhus during pregnancy, miscarriages occurred in 17% and poor neonatal outcomes in 42% of cases, which is more severe than the consequences of malaria in pregnancy [65]. Further, the mortality in patients requiring a lumbar puncture for scrub typhus CNS complications in Laos was 14% [59]. Scrub typhus is usually an easily treatable disease and the majority of these complications could be prevented by early recognition/diagnosis and increased usage of empiri- cal doxycycline [66]. It is difficult to draw any definitive conclusions from the case-fatality data due to the hetero- geneity in studies. They range from national surveillance data to case series of those admitted to ICU. National surveillance data from China, Japan and Korea provide case fatality ratios of 0.068–0.26%. However, the health facilities in these countries are significantly more advanced than other endemic countries. The fever studies from South India provide estimates of case fatality risk, but they vary from 0–33.3%—importantly, these data included patients who pre- sented to hospital and therefore will miss those that do not have severe disease. Scrub typhus disease burden protective immune responses wane over time, but detailed understanding of this remains elu- sive [61]. Moreover, the population-wide frequencies of patients with reversion to seronegativ- ity and potential disease susceptibility remain unknown, and therefore the actual exposure in these studies is likely to be substantially higher [62]. Seroprevalence Seroprevalence data was available from 5 countries only–indicating high background exposure levels, and therefore a high probability that larger numbers of unidentified and/or asymptom- atic infections occur. Disease seroprevalence data must be interpreted with caution due to unknown antibody dynamics over time and uncertainty as to whether those seropositive became sick or were asymptomatic. In scrub typhus, both humoral and cell-mediated 10 / 17 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0005838 September 25, 2017 Future focus Reports from Africa, the Middle East and most recently South America, suggest that scrub typhus is more widespread than previously appreciated. The molecular detection of Orientia spp. in rodents from Southern France and Senegal suggest that rodent-mite cycles could maintain the pathogen in nature but whether these Orientia spp. represent human pathogens is unknown [69]. The countries most affected by scrub typhus are currently experiencing profound demo- graphic, economic and ecological changes [70]. Deforestation, growing cities and climate change may lead to migration of rodents carrying infected mites and expand to more urban and non- endemic areas [8, 11, 16]. Recently the impact of an earthquake on exposing the population to the possibly perturbed soil dwelling vectors causing scrub typhus was highlighted in Nepal [71]. Ancestor et al. mapped non-malarial causes of fever, including scrub typhus, in the Mekong region [2]. Kelly et al. developed a vector map of scrub typhus based on literature review to include probable and confirmed cases that included geo-referenced locations [72]. These are useful resources that can be built upon to estimate incidence in areas where data is limited. In scrub typhus the extracted information of studies from the 1940s requires careful consider- ation to identify what data are clinically relevant today. Derne et al. summarized and mapped the distribution of rickettsia and their vectors in Oceania, confirming the widespread presence and providing a scaffold to build upon [73]. Ideally, concerted efforts in providing well main- tained up-to-date mapping of human cases and vector (chigger mite) distribution would con- tribute substantially to understanding the burden of disease. Burden of disease studies often use syndromic ‘envelopes’ for certain conditions (for exam- ple “diarrhea” or “fever”). Developing a fever ‘envelope’ approach for estimating its burden of disease, in conjunction with detailed fever etiology studies would provide improved, standard- ized and globally comparable incidence data [74, 75]. The resulting data could be stratified fur- ther and would inform on the actual burden of disease, as well as provide valuable baseline data to support economic evaluations and mathematical modeling of future interventions [76]. For example, an incentive for identifying endemic areas of scrub typhus may result in increas- ing cost-effectiveness of rapid diagnostic test (RDT) use. Testing for frequent bacterial patho- gens is likely to be economical, reducing hospitalization rates, and informs not only treatment requirements, but also appropriate antibiotic usage [77]. Scrub typhus disease burden majority of countries where scrub typhus is endemic, there is a potential bulk of relevant litera- ture that is not indexed in the databases used. The risks of publication bias and the heterogene- ity of methods and reporting in the articles limit the conclusions. Specific difficulties relating to the diagnosis of scrub typhus suggest that studies reporting data from national surveillance systems are likely to suffer from missing data due to those that do not seek medical attention are misdiagnosed or not reported. The majority of fever studies suffers from selection bias and often relies on suboptimal diagnostic tools. Study limitations This study involved an extensive search of the literature and includes up-to-date and relevant studies. However, there are several limitations; as English is not the native language in the PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0005838 September 25, 2017 11 / 17 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0005838 September 25, 2017 Acknowledgments We are grateful to Dr. Tri Wangrangsimakul for his kind support in acquiring and translating Thai epidemiological information on scrub typhus. Conceptualization: Yoel Lubell, Daniel H. Paris. Conceptualization: Yoel Lubell, Daniel H. Paris. Data curation: Ana Bonell, Paul N. Newton, John A. Crump, Daniel H. Paris. Data curation: Ana Bonell, Paul N. Newton, John A. Crump, Daniel H. Paris. Formal analysis: Ana Bonell, Yoel Lubell, Paul N. Newton, John A. Crump, Daniel H. Paris. Investigation: Ana Bonell. Formal analysis: Ana Bonell, Yoel Lubell, Paul N. Newton, John A. Crump, Daniel H. Paris. Investigation: Ana Bonell. Methodology: Ana Bonell, Daniel H. Paris. Software: Ana Bonell, Daniel H. Paris. Software: Ana Bonell, Daniel H. Paris. Supervision: Daniel H. Paris. Supervision: Daniel H. Paris. Visualization: Daniel H. Paris. Visualization: Daniel H. Paris. Writing – original draft: Ana Bonell, Daniel H. Paris. Writing – review & editing: Ana Bonell, Yoel Lubell, Paul N. Newton, John A. Crump, Daniel H. Paris. Future focus In the case of dengue, the quality of data available has improved substantially and in 2010 there were an estimated 96 million apparent and 294 million unapparent dengue infections globally [78]. Although dengue and scrub typhus both top the list of fever etiologies in multiple studies in Asia, the more easily-treatable disease is neglected–it is time for more integrated expert collaborative research to provide these urgently needed objective data [57, 78, 79]. 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https://openalex.org/W4391066589	https://jerkin.org/index.php/jerkin/article/download/188/121	Indonesian	null	Pelaksanaan Sekolah Perempuan untuk Memberikan Pemahaman tentang Relationship and Sex Education	Jurnal Pengabdian Masyarakat dan Riset Pendidikan	2,023	cc-by-sa	3,227	Pelaksanaan Sekolah Perempuan untuk Memberikan Pemahaman tentang Relationship and Sex Education Cherish Mauretha Mahar Violyn1*, Afifah Fitri Desyana2, Jonanda Wahyu Mahendra3, Zahrandy Bintang Bhaskara Argono4, Vera Arista Aprilia5, Amy Nurlitasari6, Deaningma Maharani7, Klarisa Afif Nusaibah8, Vera Putri Meytasharoh9, Clera Agretyas Nur Anisa10, Ardhana Januar Mahardhani11 Cherish Mauretha Mahar Violyn1 , Afifah Fitri Desyana2, Jonanda Wahyu Mahendra3, Zahrandy Bintang Bhaskara Argono4, Vera Arista Aprilia5, Amy Nurlitasari6, Deaningma Maharani7, Klarisa Afif Nusaibah8, Vera Putri Meytasharoh9, Clera Agretyas Nur Anisa10, Ardhana Januar Mahardhani11 1-11Universitas Muhammadiyah Ponorogo, Jl. Budi Utomo No.10, Ronowijayan, Kec. Ponorogo, Kabupaten Ponorogo, Jawa Timur 63471 E-mail: cherishmmv@gmail.com https://doi.org/10.31004/jerkin.v2i2.188 y , y , y , Zahrandy Bintang Bhaskara Argono4, Vera Arista Aprilia5, Amy Nurlitasari6, Deaningma Maharani7, Klarisa Afif Nusaibah8, Vera Putri Meytasharoh9, Clera Agretyas Nur Anisa10, Ardhana Januar Mahardhani11 1-11Universitas Muhammadiyah Ponorogo, Jl. Budi Utomo No.10, Ronowijayan, Kec. Ponorogo, Kabupaten Ponorogo, Jawa Timur 63471 E-mail: cherishmmv@gmail.com https://doi.org/10.31004/jerkin.v2i2.188 Keberadaan Sekolah Perempuan Dewi Songgolangit di Desa Lembah Kecamatan Babadan Kabupaten Ponorogo dapat memberikan alternatif penyelesaian dari maraknya isu dispensasi pernikahan dan kehamilan pra- nikah yang ada di Kabupaten Ponorogo. Dari hasil observasi dan wawancara diketahui bahwa Desa Lembah merupakan salah satu desa yang banyak mengirimkan Pekerja Migran Indonesia (PMI). Ketidakhadiran orang tua terutama ibu dalam perkembangan anak perempuannya menjadi masalah sendiri yang harus diselesaikan. Oleh karenanya, Sekolah Perempuan Dewi Songgolangit ini menerapkan kurikulum Relationship and Sex Education (RSE) yang disesuaikan dengan jenjang usia sasaran. Kegiatan ini diikuti oleh 60 anak yang dibagi menjadi tiga kelas, yaitu kelas A untuk anak usia 9 – 12 tahun sebanyak 25 anak, kelas B untuk anak usia 13 – 15 tahun sebanyak 20 anak, dan kelas C untuk anak usia 16 – 18 tahun sebanyak 15 anak. Metode yang digunakan dalam kegiatan Sekolah Perempuan ini adalah penyampaian materi, diskusi, serta presentasi. Dari hasil kegiatan yang dilaksanakan dapat dilihat indikator keberhasilan program yaitu meningkatnya pemahaman siswa Sekolah Perempuan Dewi Songgolangit sebesar 82,84%. Keywords: All girls' school, Relationships and Sex Education. The existence of the Dewi Songgolangit Women's School in Lembah Village, Babadan District, Ponorogo Regency can provide an alternative solution to the widespread issue of marriage dispensation and married by accident in Ponorogo Regency. From the results of observations and interviews, it is known that Lembah Village is one of the villages that sends many Indonesian Migrant Workers (PMI). The absence of parents, especially mothers, in the development and growth of their daughters is a problem itself that must be resolved. p-ISSN: 2963-7856 \| e-ISSN: 2961-9890 Available online at jerkin.org/index.php/jerkin Jurnal Pengabdian Masyarakat dan Riset Pendidikan Volume 2 No 2, Oktober-Desember 2023, pp 01-06 How to Cite: Violyn et al. (2023). Pelaksanaan Sekolah Perempuan untuk Memberikan Pemahaman tentang Relationship and Sex Education, 2(2) 01-06. https://doi.org/10.31004/jerkin.v2i2.188 PENDAHULUAN Permasalahan tentang dispensasi pernikahan menjadi isu yang terus berkembang di Kabupaten Ponorogo (Rosa, 2023; Ulfah, 2023), banyak hal yang mempengaruhi adanya dispensasi pernikahan diantaranya adalah masalah ekonomi yang mengharuskan mereka menikah muda, hamil diluar nikah, dampak sosial ekonomi dari Covid-19 (Mahardhani, 2020), serta masalah sosial lain yang disebabkan ketidakhadiran orang tua terutama ibu sebagai pendamping kehidupan para remaja. Masalah ini tidak bisa dibiarkan begitu saja, sehingga orang tua harus selalu mengontrol keberadaan anaknya, lingkungan pertemanan dan permainan, sampai dengan apa yang mereka lakukan ketika berada di luar rumah. Hal ini perlu mendapatkan perhatian khusus terutama untuk anak perempuan yang sedang berada dalam masa pertumbuhan dan penemuan jati diri. p p j Ketidakhadiran orang tua terutama ibu menjadi permasalahan sendiri yang harus dipecahkan. Ibu yang harusnya selalu mendampingi anak perempuannya untuk bertumbuh tetapi justru ditinggalkan untuk bekerja ke luar kota dan luar negeri (Amalia, 2011). Hal ini tentu mengakibatkan pola asuh yang seharusnya bersama orang tua diserahkan kepada kakek-nenek atau suadara dari ayah atau ibunya. entu ada banyak aktivitas yang tidak bisa dikontrol dan mengakibatkan banyaknya kasus- kasus tentang pernikahan muda atau hamil di luar nikah. Bagi remaja yang memiliki ibu sebagai pekerja migran, dinamika psikologis yang terjadi dalam proses perkembangan anak akan tidak dapat sempurna karena ketidakhadiran tokoh sentral sebagai figur mereka. Kepribadian remaja akan berkembang dengan baik melalui pembiasaan sejak dini dalam keluarga. Keluarga merupakan lingkungan pertama dan utama yang mempengaruhi perkembangan kepribadian anak. Remaja yang tinggal dalam keluarga yang mampu memenuhi kebutuhan biologis, psikologis, dan sosialnya akan tumbuh dan berkembang secara sehat, mengembangkan potensi dirinya dan belajar memecahkan masalah dan tanggung jawab. Hal ini ditunjukkan melalui harga diri (self-esteem) yang positif dari seorang remaja (Amalia, 2011). Salah satu desa yang banyak terdapat pekerja migran tersebut adalah Desa Lembah di Kecamatan Babadan Kabupaten Ponorogo, dari data BPS jumlah penduduk di Desa Lembah adalah 4.993 jiwa atau 1.642 kepala keluarga (KK) dengan rincian jumlah penduduk laki-laki sebanyak 2.467 jiwa dan jumlah penduduk perempuan sebanyak 2.526 jiwa. Penduduk Desa Lembah sebagian besar bekerja menjadi petani dan bekerja di luar negeri sebagai Pekerja Migran Indonesia (PMI). Hal ini sesuai dengan yang disampaikan oleh bapak Kepala Desa Lembah, Bapak Heri Setyo Kurniawan. PENDAHULUAN Oleh karena itulah tim pelaksana PPK Ormawa BEM FKIP Universitas Muhammadiyah Ponorogo memilih Desa Lembah untuk melakukan kegiatan Sekolah Perempuan ini harapannya para anak perempuan yang orangtuanya bekerja sebagai PMI dapat mempelajari tentang konsep dasar keluarga, cara berhubungan yang baik (pertemanan, perundungan, percintaan, pernikahan, dan perceraian), mengenal tentang bagian tubuh dan perubahannya, memahami dan membedakan jenis-jenis perasaan (kebahagiaan dan kesedihan, perubahan perasaan, mimpi basah, jatuh cinta, dan menghadapi konflik dalam percintaan), mengetahui proses reproduksi manusia, mengetahui cara menjaga dan merawat diri, memahami perilaku seksual di masyarakat dan hukum yang mengaturnya, juga mengenal orang-orang di sekitar yang dapat dimintai bantuan dalam kondisi tertentu. Siswa dalam Sekolah perempuan ini juga akan dikenalkan dengan beberapa aplikasi kesehatan seksual yang dapat diakses dan diunduh secara gratis di internet. Melalui program sekolah perempuan ini, permasalahan kurangnya edukasi seksual di kalangan anak perempuan pekerja migran Indonesia dapat ditekan dengan mendapatkan wadah untuk belajar dan bertanya tentang keluarga dan seksualitas tanpa takut dicap tabu dan kurang sopan. Pelaksanaan Sekolah Perempuan untuk Memberikan Pemahaman tentang Relationship and Sex Education Therefore, the Dewi Songgolangit Women's School implements a Relationship and Sex Education (RSE) curriculum that is adapted to the target age level. This activity was attended by 60 children who were divided into three classes, namely class A for children aged 9 - 12 years with 25 children, class B for children aged 13 - 15 with 20 children, and class C for children aged 16 - 18 years with 15 children. The methods used in this Women's School activity are delivery of material, discussions and presentations. From the results of the activities carried out, indicators of the success of the program can be seen, namely the increase the understanding of Dewi Songgolangit Women's School student by 82.84%. How to Cite: Violyn et al. (2023). Pelaksanaan Sekolah Perempuan untuk Memberikan Pemahaman tentang Relationship and Sex Education, 2(2) 01-06. https://doi.org/10.31004/jerkin.v2i2.188 1 1 Jurnal Pengabdian Masyarakat dan Riset Pendidikan, Volume 2, No. 2, Oktober-Desember 2023, hal. 01-06 2 Copyright © 2023, Jurnal Pengabdian Masyarakat dan Riset Pendidikan ISSN 2963-7856 (print), ISSN 2961-9890 (online) METODE Kegiatan Sekolah Perempuan ini dinamakan Sekolah Perempuan Dewi Songgolangit. Sekolah Perempuan Dewi Songgolangit dilaksanakan di Desa Lembah, Kecamatan Babadan, Kabupaten Ponorogo. Pelaksanaan seluruh kegiatan dilakukan selama 12 kali pertemuan setiap hari Sabtu dan Minggu pada bulan September dan Oktober tahun 2023. Kegiatan ini diikuti oleh 60 anak yang dibagi menjadi tiga kelas, yaitu kelas A untuk anak usia 8 – 11 tahun sebanyak 25 anak, kelas B untuk anak usia 12 – 15 tahun sebanyak 20 anak, dan kelas C untuk anak usia 16 – 18 tahun sebanyak 15 anak. Metode yang digunakan dalam kegiatan Sekolah Perempuan ini adalah penyampaian materi, diskusi, Pelaksanaan Sekolah Perempuan untuk Memberikan Pemahaman tentang Relationship and Sex Education, Cherish Mauretha Mahar Violyn, Afifah Fitri Desyana, Jonanda Wahyu Mahendra, Zahrandy Bintang Bhaskara Argono, Vera Arista Aprilia, Amy Nurlitasari, Deaningma Maharani, Klarisa Afif Nusaibah, Vera Putri Meytasharoh , Clera Agretyas Nur Anisa, Ardhana Januar Mahardhani 3 serta presentasi. Adapun tahapan yang dilakukan dalam Sekolah Perempuan Dewi Songgolangit ini adalah: Pelaksanaan Sekolah Perempuan untuk Memberikan Pemahaman tentang Relationship and Sex Education, Cherish Mauretha Mahar Violyn, Afifah Fitri Desyana, Jonanda Wahyu Mahendra, Zahrandy Bintang Bhaskara Argono, Vera Arista Aprilia, Amy Nurlitasari, Deaningma Maharani, Klarisa Afif Nusaibah, Vera Putri Meytasharoh , Clera Agretyas Nur Anisa, Ardhana Januar Mahardhani 3 serta presentasi. Adapun tahapan yang dilakukan dalam Sekolah Perempuan Dewi Songgolangit ini adalah: Pelaksanaan Sekolah Perempuan untuk Memberikan Pemahaman tentang Relationship and Sex Education, Cherish Mauretha Mahar Violyn, Afifah Fitri Desyana, Jonanda Wahyu Mahendra, Zahrandy Bintang Bhaskara Argono, Vera Arista Aprilia, Amy Nurlitasari, Deaningma Maharani, Klarisa Afif Nusaibah, Vera Putri Meytasharoh , Clera Agretyas serta presentasi. Adapun tahapan yang dilakukan dalam Sekolah Perempuan Dewi Songgol adalah: 1. perencanaan, 2. pelaksanaan, dan 3. evaluasi dan rencana tindak lanjut. HASIL DAN DISKUSI Kegiatan Sekolah Perempuan Dewi Songgolangit ini tidak dilaksanakan secara mandiri oleh tim pelaksana, tetapi tim pelaksana mengajak berbagai elemen untuk bekerjasama dalam rangka menjadi pakar yang akan melaksanakan pembelajaran, diantaranya berasal dari kelompok Pusat Informasi dan Konseling Remaja (PIK-R) Fajar Melati, Duta Genre Kabupaten Ponorogo, psikolog, dosen, serta tenaga kesehatan dari Universitas Muhammadiyah Ponorogo. Adapun kegiatan dalam Sekolah Perempuan Dewi Songgolangit ini dapat diuraikan sebagai berikut: a. Perencanaan a. Perencanaan Perencanaan diawali dengan koordinasi yang dilakukan dengan Pemerintah Desa Lembah dan Karang Taruna setempat. Berdasarkan observasi dan wawancara dengan Kepala Desa Lembah, Bapak Heri Setyo Kurniawan, diketahui bahwa Desa Lembah memiliki jumlah anak perempuan dari pekerja migran Indonesia sejumlah 60 anak. Anak- anak dan remaja perempuan tersebut dinilai memiliki potensi melangsungkan pernikahan dini yang disebabkan karena hamil diluar nikah atau kurangnya kesadaran akan pentingnya pendidikan sehingga memilih untuk putus sekolah. Sehingga, dalam program Sekolah Perempuan ini, tim PPK Ormawa BEM FKIP yang bekerjasama dengan Pemerintah Desa Lembah, berinisiatif untuk memberikan sosialisasi Relationship and Sex Education (RSE) yang tidak sempat mereka dapatkan karena tidak adanya pendampingan langsung dari orang tua. g Setelah koordinasi dilaksanakan, tim pelaksana melakukan pendataan jumlah peserta yang akan mengikuti Sekolah Perempuan Dewi Songgolangit. Dalam tahap perencanaan ini juga dibuat kurikulum yang akan diimplementasikan pada Sekolah Perempuan Dewi Songgolangit. Kurikulum ini mengacu pada panduan International Technical Guidence-Informed Approach – revision edition (2018) oleh UNESCO dan Curriculum Design Tool for Relationship and Sex Education (2018) oleh Sex Education Forum the Charity Commision for England and Wales. Tentunya kurikulum yang disusun oleh tim pelaksana ini telah disesuaikan dengan keadaan dan lokalitas Kabupaten Ponorogo. Oleh karena itu, pembelajaran yang dilakukan bersifat kontekstual dan lebih mudah dibayangkan oleh peserta didik karena dekat dengan aktivitas keseharian. Salah satu kegiatan yang termasuk dalam perencanaan pembuatan kurikulum ini adalah Focus Group Discussion (FGD) yang telah dilaksanakan pada tanggal 30 Agustus 2023 (Violyn et al., 2023) b. Kegiatan Sekolah Perempuan Dewi Songgolangit ini dilaksanakan selama 12 kali pertemuan yang dibagi menjadi 1 kali pertemuan orientasi dan pre test, 10 kali pertemuan pemaparan materi, dan 1 kali pertemuan penutupan dan post test. Salah satu indikator keberhasilan untuk mengukur keberhasilan program yaitu meningkatnya nilai pre-test dan post-test sebesar 80% yang selanjutnya akan dibahas dalam bagian evaluasi dan rencana tindak lanjut. g j Adapun materi yang disampaikan dalam 10 kali pertemuan terdapat dalam tabel 1. Tabel 1. Jadwal Kegiatan dan Materi Pertemuan ke- Tanggal Materi Pembelajaran Pengajar 1. 9 September 2023 Relationship: Keluarga Dwi Estiningsih, M.Psi., Psikolog. 2. 10 September 2023 Relationship: Pertemanan dan Hubungan Romantis Anindhea Firdausi Azzahra. 3. 16 September 2023 Organ Reproduksi dan Perkembangannya: Pubertas Wahyu Ari Ike Lestariningtyas 4. 17 September 2023 Organ Reproduksi dan Perkembangannya: Menstruasi Aprilia Eka Indarti Tabel 1. Jadwal Kegiatan dan Materi Jurnal Pengabdian Masyarakat dan Riset Pendidikan, Volume 2, No. 2, Oktober-Desember 2023, hal. Copyright © 2023, Jurnal Pengabdian Masyarakat dan Riset Pendidikan ISSN 2963-7856 (print), ISSN 2961-9890 (online) a. Perencanaan 01-06 4 Pertemuan ke- Tanggal Materi Pembelajaran Pengajar 5. 23 September 2023 Seksualitas dan Kesehatan Reproduksi: Infeksi Menular Seksual Klarisa Afif Nusaibah 6. 24 September 2023 Seksualitas dan Kesehatan Reproduksi: HIV dan AIDS Cherish Mauretha Mahar Violyn 7. 30 September 2023 Nilai, Hak, dan Sekualitas: Hak-hak anak Hadi Cahyono, M.Pd 8. 1 Oktober 2023 Nilai, Hak, dan Sekualitas: Nilai sosial dalam masyarakat dan seksualitas Hadi Cahyono, M.Pd. 9. 7 Oktober 2023 Kekerasan dan Pencegahannya: Pelecahan Seksual Hadi Cahyono, M.Pd. 10. 8 Oktober 2023 Kekerasan dan Pencegahannya: Penggunaan Media Sosial yang Aman Clera Agretyas Nur Anisa Jurnal Pengabdian Masyarakat dan Riset Pendidikan, Volume 2, No. 2, Oktober-Desember 2023, hal. 01-06 4 Kegiatan ini dilaksanakan dengan berbagai metode pembelajaran, hal ini dimaksudkan untuk meningkatkan semangat dan pemahaman para peserta didik baik di kelas A, B, dan C. Kegiatan ini dilaksanakan dengan berbagai metode pembelajaran, hal ini dimaksudkan untuk meningkatkan semangat dan pemahaman para peserta didik baik di kelas A, B, dan C. Gambar 1. Pembelajaran tentang Nilai, Hak, dan Seksualitas Gambar 2. Presentasi tentang Cyberbullying c. Evaluasi dan Rencana Tindak Lanjut Evaluasi dilakukan oleh tim pelaksana, Ormawa BEM FKIP, dan pihak Perguruan Tinggi terhadap seluruh pelaksanaan program yaitu dari pertemuan pertama sampai dengan pertemuan keduabelas. Dalam hal ini, Ormawa BEM FKIP melakukan evaluasi proses kegiatan (process evaluation) dan Perguruan Tinggi melakukan evaluasi dampak (outcomes evaluation) di akhir kegiatan dengan melakukan monitoring dan evaluasi dengan pakar sebagai tim penilai. Evaluasi ini juga dilaksanakan sebagai upaya perbaikan dari tim terhadap pelaksanaan sebelumnya. Selain itu, tim pelaksana juga melakukan evaluasi kegiatan peserta didik melalui isian kuesioner, hal ini untuk mengetahui kemampuan anak yang ditunjukkan peningkatan nilai dari pre-test dan post-test, seperti yang dapat dilihat dalam tabel 2 Gambar 1. Pembelajaran tentang Nilai, Hak, dan Seksualitas Gambar 1. Pembelajaran tentang Nilai, Hak, dan Seksualitas Gambar 2 Presentasi tentang Cyberbullying Gambar 2. Presentasi tentang Cyberbullying c. Evaluasi dan Rencana Tindak Lanjut c. Evaluasi dan Rencana Tindak Lanjut Evaluasi dilakukan oleh tim pelaksana, Ormawa BEM FKIP, dan pihak Perguruan Tinggi terhadap seluruh pelaksanaan program yaitu dari pertemuan pertama sampai dengan pertemuan keduabelas. Dalam hal ini, Ormawa BEM FKIP melakukan evaluasi proses kegiatan (process evaluation) dan Perguruan Tinggi melakukan evaluasi dampak (outcomes evaluation) di akhir kegiatan dengan melakukan monitoring dan evaluasi dengan pakar sebagai tim penilai. Evaluasi ini juga dilaksanakan sebagai upaya perbaikan dari tim terhadap pelaksanaan sebelumnya. a. Perencanaan Selain itu, tim pelaksana juga melakukan evaluasi kegiatan peserta didik melalui isian kuesioner, hal ini untuk mengetahui kemampuan anak yang ditunjukkan peningkatan nilai dari pre-test dan post-test, seperti yang dapat dilihat dalam tabel 2. c. Evaluasi dan Rencana Tindak Lanjut Evaluasi dilakukan oleh tim pelaksana, Ormawa BEM FKIP, dan pihak Perguruan Tinggi terhadap seluruh pelaksanaan program yaitu dari pertemuan pertama sampai dengan pertemuan keduabelas. Dalam hal ini, Ormawa BEM FKIP melakukan evaluasi proses kegiatan (process evaluation) dan Perguruan Tinggi melakukan evaluasi dampak (outcomes evaluation) di akhir kegiatan dengan melakukan monitoring dan evaluasi dengan pakar sebagai tim penilai. Evaluasi ini juga dilaksanakan sebagai upaya perbaikan dari tim terhadap pelaksanaan sebelumnya. Selain itu, tim pelaksana juga melakukan evaluasi kegiatan peserta didik melalui isian kuesioner, hal ini untuk mengetahui kemampuan anak yang ditunjukkan peningkatan nilai dari pre-test dan post-test, seperti yang dapat dilihat dalam tabel 2. Copyright © 2023, Jurnal Pengabdian Masyarakat dan Riset Pendidikan ISSN 2963-7856 (print), ISSN 2961-9890 (online) Pelaksanaan Sekolah Perempuan untuk Memberikan Pemahaman tentang Relationship and Sex Education, Cherish Mauretha Mahar Violyn, Afifah Fitri Desyana, Jonanda Wahyu Mahendra, Zahrandy Bintang Bhaskara Argono, Vera Arista Aprilia, Amy Nurlitasari, Deaningma Maharani, Klarisa Afif Nusaibah, Vera Putri Meytasharoh , Clera Agretyas Nur Anisa, Ardhana Januar Mahardhani 5 Tabel 2. Perhitungan N-Gain Score Sekolah Perempuan Pre-Test Post-Test Post-Pre Score Ideal N-Gain Score N-Gain Score % Mean 54,875 91,812 36,937 45,125 0,83 82,84 Pelaksanaan Sekolah Perempuan untuk Memberikan Pemahaman tentang Relationship and Sex Education, Cherish Mauretha Mahar Violyn, Afifah Fitri Desyana, Jonanda Wahyu Mahendra, Zahrandy Bintang Bhaskara Argono, Vera Arista Aprilia, Amy Nurlitasari, Deaningma Maharani, Klarisa Afif Nusaibah, Vera Putri Meytasharoh , Clera Agretyas Nur Anisa, Ardhana Januar Mahardhani 5 elaksanaan Sekolah Perempuan untuk Memberikan Pemahaman tentang elationship and Sex Education, Cherish Mauretha Mahar Violyn, Afifah Fitri Desyana, 5 Tabel 2. Perhitungan N-Gain Score Sekolah Perempuan Dari tabel 2 dapat dilihat bahwa adanya kegiatan Sekolah Perempuan Dewi Songgolangit dapat memberikan peningkatan pengetahuan remaja putri Desa Lembah terhadap relationship and sex education. Melalui diadakannya pre-test dan post-test, dapat diketahui hasil peningkatan ketrampilan proses sains dan hasil belajar kognitif antara sebelum diberikan pemahaman materi dan sesudah pemahaman materi adalah sebesar 82,84%. Selanjutnya, rencana tindak lanjut kegiatan juga dilakukan oleh tim dalam rangka menjaga keberlanjutan program. Program ini tidak berhenti begitu saja setelah dua belas kali pertemuan selesai dilaksanakan. Namun, kegiatan diskusi dan penyampaian materi akan terus dilakukan melalui whatssapp group chat yang telah dibentuk diawal pertemuan. a. Perencanaan Pelaksanaan tindaklanjut program sudah dilakukan pada tanggal 15 Oktober 2023 dengan melibatkan unsur-unsur lain seperti pihak Perguruan Tinggi, BEM FKIP, tim pelaksana PPK Ormawa, Pemerintah desa, serta Karang Taruna dari Dusun Ngijo dan Jajar. Gambar 3. Evaluasi kegiatan dan penyampaian tindak lanjut Gambar 3. Evaluasi kegiatan dan penyampaian tindak lanjut Copyright © 2023, Jurnal Pengabdian Masyarakat dan Riset Pendidikan ISSN 2963-7856 (print), ISSN 2961-9890 (online) Jurnal Pengabdian Masyarakat dan Riset Pendidikan, Volume 2, No. 2, Oktober-Desember 2023, hal. 01-06 Ulfah, I. (2023, January 16). Ratusan Siswa di Ponorogo Hamil di Luar Nikah, Benarkah? Mari Cek Faktanya!! . Https://Iainponorogo.Ac.Id. https://iainponorogo.ac.id/2023/01/16/ratusan-siswa- di-ponorogo-hamil-di-luar-nikah-benarkah-mari-cek-faktanya/ KESIMPULAN Kegiatan Sekolah Perempuan Dewi Songgolangit ini menjadi salah satu solusi yang diberikan oleh Tim PPK Ormawa BEM FKIP kepada Pemerintah Desa Lembah dalam rangka memberikan alternatif penyelesaian terhadap munculnya isu dispensasi pernikahan dini dan kehamilan pra nikah. Sekolah Perempuan Dewi Songgolangit ini mengusung tema Relationship and Sex Education yang berisikan tentang pemahaman tentang relationship, organ reproduksi dan perkembangannya, seksualitas dan kesehatan reproduksi, nilai, hak, dan seksualitas, serta kekerasan dan pencegahannya. Dari hasil kegiatan yang dilaksanakan dapat dilihat indikator keberhasilan program yaitu meningkatnya pemahaman siswa Sekolah Perempuan Dewi Songgolangit yaitu 82,84%. REFERENSI Amalia, L. (2011). Dampak Ketidakhadiran Ibu Sebagai Tenaga Kerja Wanita (TKW) Terhadap Perkembangan Psikologis Remaja. Kodifikasia, 5(1), 79–96. https://doi.org/10.21154/KODIFIKASIA.V5I1.752 Mahardhani, A. J. (2020). Menjadi Warga Negara yang Baik pada Masa Pandemi Covid-19: Persprektif Kenormalan Baru. Jurnal Pancasila Dan Kewarganegaraan. https://doi.org/10.24269/jpk.v5.n2.2020.pp65-76 p g y Violyn, C. M. M., Desyana, A. F., Mahendra, J. W., Argono, Z. B. B., Aprilia, V. A., Nurlitasari, A., Maharani, D., Nusaibah, K. A., Meytasharoh, V. P., Anisa, C. A. N., & Mahardhani, A. J. (2023). Pelaksanaan Focus Group Discussion untuk Penyiapan Materi Relationship and Sex Education pada Pelaksanaan Sekolah Perempuan. Jurnal Pengabdian Nasional (JPN) Indonesia, 4(3), 594–600. https://doi.org/10.35870/JPNI.V4I3.456 tanggapan pakar Ulfah, I. (2023, January 16). Ratusan Siswa di Ponorogo Hamil di Luar Nikah, Benarkah? Mari Cek Faktanya!! . Https://Iainponorogo.Ac.Id. https://iainponorogo.ac.id/2023/01/16/ratusan-siswa- di-ponorogo-hamil-di-luar-nikah-benarkah-mari-cek-faktanya/ Violyn, C. M. M., Desyana, A. F., Mahendra, J. W., Argono, Z. B. B., Aprilia, V. A., Nurlitasari, A., Maharani, D., Nusaibah, K. A., Meytasharoh, V. P., Anisa, C. A. N., & Mahardhani, A. J. (2023). Pelaksanaan Focus Group Discussion untuk Penyiapan Materi Relationship and Sex Education pada Pelaksanaan Sekolah Perempuan. Jurnal Pengabdian Nasional (JPN) Indonesia, 4(3), 594–600. https://doi.org/10.35870/JPNI.V4I3.456 Jurnal Pengabdian Masyarakat dan Riset Pendidikan, Volume 2, No. 2, Oktober-Desember 2023, hal. 01-06 6 Rosa, N. (2023, January 17). Serba-serbi Ratusan Anak di Ponorogo Minta Dispensasi Nikah: Alasan hingga Tanggapan Pakar. Https://Www.Detik.Com. https://www.detik.com/edu/detikpedia/d- 6520471/serba-serbi-ratusan-anak-di-ponorogo-minta-dispensasi-nikah-alasan-hingga- tanggapan-pakar Rosa, N. (2023, January 17). Serba-serbi Ratusan Anak di Ponorogo Minta Dispensasi Nikah: Alasan hingga Tanggapan Pakar. Https://Www.Detik.Com. https://www.detik.com/edu/detikpedia/d- 6520471/serba-serbi-ratusan-anak-di-ponorogo-minta-dispensasi-nikah-alasan-hingga- tanggapan-pakar Ulfah, I. (2023, January 16). Ratusan Siswa di Ponorogo Hamil di Luar Nikah, Benarkah? Mari Cek Faktanya!! . Https://Iainponorogo.Ac.Id. https://iainponorogo.ac.id/2023/01/16/ratusan-siswa- di-ponorogo-hamil-di-luar-nikah-benarkah-mari-cek-faktanya/ Violyn, C. M. M., Desyana, A. F., Mahendra, J. W., Argono, Z. B. B., Aprilia, V. A., Nurlitasari, A., Maharani, D., Nusaibah, K. A., Meytasharoh, V. P., Anisa, C. A. N., & Mahardhani, A. J. (2023). Pelaksanaan Focus Group Discussion untuk Penyiapan Materi Relationship and Sex Education pada Pelaksanaan Sekolah Perempuan. Jurnal Pengabdian Nasional (JPN) Indonesia, 4(3), 594–600. https://doi.org/10.35870/JPNI.V4I3.456
https://openalex.org/W3158823894	https://digital.library.adelaide.edu.au/dspace/bitstream/2440/134537/2/hdl_134537.pdf	English	null	Search for <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:mrow><mml:msup><mml:mrow><mml:mi>B</mml:mi></mml:mrow><mml:mrow><mml:mo>+</mml:mo></mml:mrow></mml:msup><mml:mo stretchy="false">→</mml:mo><mml:msup><mml:mrow><mml:mi>K</mml:mi></mml:mrow><mml:mrow><mml:mo>+</mml:mo></mml:mrow></mml:msup><mml:mi>ν</mml:mi><mml:mover accent="true"><mml:mrow><mml:mi>ν</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy="false">¯</mml:mo></mml:mrow></mml:mover></mml:mrow></mml:math…	Physical review letters	2,021	cc-by	8,402	PHYSICAL REVIEW LETTERS 127, 181802 (2021) PHYSICAL REVIEW LETTERS 127, 181802 (2021) Search for B + →K + ν¯ν Decays Using an Inclusive Tagging Method at Belle II Search for B + →K + ν¯ν Decays Using an Inclusive Tagging Method at Belle II F. Abudin´en,44 I. Adachi,21,19 K. Adamczyk,66 P. Ahlburg,97 H. Aihara,113 N. Akopov,119 A. Aloisio,86,37 N. Anh Ky,34,12 D. M. Asner,2 H. Atmacan,99 T. Aushev,23 V. Aushev,79 A. Baur,10 V. Babu,10 S. Baehr,48 P. Bambade,94 Sw. Banerjee,103 S. Bansal,72 J. Baudot,95 J. Becker,48 P. K. Behera,27 J. V. Bennett,107 E. Bernieri,42 F. U. Bernlochner,97 M. Bertemes,30 E. Bertholet,82 M. Bessner,101 S. Bettarini,89,40 F. Bianchi,91,43 T. Bilka,6 D. Biswas,103 A. Bozek,66 M. Bračko,105,78 P. Branchini,42 N. Braun,48 T. E. Browder,101 A. Budano,42 S. Bussino,90,42 M. Campajola,86,37 L. Cao,97 G. Casarosa,89,40 C. Cecchi,88,39 D. Červenkov,6 P. Chang,65 R. Cheaib,10 V. Chekelian,59 C. Chen,49 Y.-T. Chen,65 B. G. Cheon,20 K. Chilikin,54 K. Chirapatpimol,7 K. Cho,51 S.-J. Cho,120 S. Choudhury,26 D. Cinabro,117 L. Corona,89,40 L. M. Cremaldi,107 S. Cunliffe,10 T. Czank,114 F. Dattola,10 E. De La Cruz-Burelo,5 G. de Marino,94 G. De Nardo,86,37 M. De Nuccio,10 G. De Pietro,42 R. de Sangro,36 M. Destefanis,91,43 S. Dey,82 A. De Yta-Hernandez,5 A. Di Canto,2 F. Di Capua,86,37 J. Dingfelder,97 Z. Doležal,6 I. Domínguez Jim´enez,85 T. V. Dong,12 K. Dort,47 S. Dubey,101 S. Duell,97 G. Dujany,95 S. Eidelman,3,54,68 M. Eliachevitch,97 D. Epifanov,3,68 T. Ferber,10 D. Ferlewicz,106 T. Fillinger,95 G. Finocchiaro,36 S. Fiore,41 A. Fodor,60 F. Forti,89,40 A. Frey,17 B. G. Fulsom,71 N. Gabyshev,3,68 E. Ganiev,92,44 M. Garcia-Hernandez,5 A. Garmash,3,68 V. Gaur,116 A. Gaz,87,38 A. Gellrich,10 R. Giordano,86,37 A. Giri,26 A. Glazov,10 B. Gobbo,44 R. Godang,111 P. Goldenzweig,48 B. Golob,102,78 P. Grace,96 W. Gradl,46 E. Graziani,42 D. Greenwald,81 Y. Guan,99 K. Gudkova,3,68 C. Hadjivasiliou,71 S. Halder,80 K. Hara,21,19 O. Hartbrich,101 K. Hayasaka,67 H. Hayashii,64 S. Hazra,80 C. Hearty,98,33 I. Heredia de la Cruz,5,9 M. Hernández Villanueva,107 A. Hershenhorn,98 T. Higuchi,114 E. C. Hill,98 H. Hirata,62 M. Hoek,46 M. Hohmann,106 C.-L. Hsu,112 T. Humair,59 T. Iijima,62,63 K. Inami,62 G. Inguglia,30 J. Irakkathil Jabbar,48 A. Ishikawa,21,19 R Itoh 21,19 M Iwasaki 69 Y Iwasaki 21 P Jackson 96 W W Jacobs 28 D E Jaffe 2 Y Jin 44 C Joo 114 H Junkerkalefeld 97 Search for B + →K + ν¯ν Decays Using an Inclusive Tagging Method at Belle II F. Abudin´en,44 I. Adachi,21,19 K. Adamczyk,66 P. Ahlburg,97 H. Aihara,113 N. Akopov,119 A. Aloisio,86,37 N. Anh Ky,34,12 D. M. Asner,2 H. Atmacan,99 T. Aushev,23 V. Aushev,79 A. Baur,10 V. Babu,10 S. Baehr,48 P. Bambade,94 Sw. Banerjee,103 S. Bansal,72 J. Baudot,95 J. Becker,48 P. K. Search for B + →K + ν¯ν Decays Using an Inclusive Tagging Method at Belle II Schueler,101 C. Schwanda,30 A. J. Schwartz,99 181802-1 Published by the American Physical Society 0031-9007=21=127(18)=181802(10) 181802-1 Search for B + →K + ν¯ν Decays Using an Inclusive Tagging Method at Belle II Lautenbach,1 F. R. Le Diberder,94 S. C. Lee,53 P. Leitl, D. Levit, P. M. Lewis, C. Li, L. K. Li, S. X. Li, Y. B. Li, J. Libby, K. Lieret, Z. Liptak, Q. Y. Liu, D. Liventsev,117,21 S. Longo,10 A. Lozar,78 T. Lueck,56 C. Lyu,97 M. Maggiora,91,43 S. Maity,25 R. Manfredi,92,44 E. Manoni,39 D. Liventsev,117,21 S. Longo,10 A. Lozar,78 T. Lueck,56 C. Lyu,97 M. Maggiora,91,43 S. Maity,25 R. Manfredi,92,44 E. Manoni,39 S. Marcello,91,43 C. Marinas,35 A. Martini,10 M. Masuda,14,70 T. Matsuda,108 K. Matsuoka,21 D. M 86 37 48 106 115 64 21 19 M. Merola,86,37 F. Metzner,48 M. Milesi,106 C. Miller,115 K. Miyabayashi,64 H. Miyake,21,19 R. Mizuk,54,23 G. B. Mohanty,80 H.-G. Moser,59 M. Mrvar,30 F. J. Müller,10 C. Murphy,114 R. Mussa,43 K. R. Nakamura,21,19 M. 101 82 119 10 2 21 19 Mrvar,30 F. J. Müller,10 C. Murphy,114 R. Mussa,43 K. R. Nakamura,21,19 M. Nakao,21,19 Z. Natkan 82 119 10 2 21 19 101 A. Natochii,101 M. Nayak,82 G. Nazaryan,119 C. Niebuhr,10 N. K. Nisar,2 S. Nishida,21,19 K. Nishimura,101 S. Ogawa,83 Y. Onishchuk,79 H. Ono,67 Y. Onuki,113 P. Oskin,54 H. Ozaki,21,19 P. Pakhlov,54,61 G. Pakhlova,23,54 A. Paladino,89,40 109 107 89 40 37 53 21 97 42 103 A. Natochii,101 M. Nayak,82 G. Nazaryan,119 C. Niebuhr,10 N. K. Nisar,2 S. Nishida,21,19 K. Nishimura,101 S. Ogawa,83 Y. Onishchuk,79 H. Ono,67 Y. Onuki,113 P. Oskin,54 H. Ozaki,21,19 P. Pakhlov,54,61 G. Pakhlova,23,54 A. Paladino,89,40 T. Pang,109 A. Panta,107 E. Paoloni,89,40 S. Pardi,37 H. Park,53 S.-H. Park,21 B. Paschen,97 A. Passeri,42 A. Pathak,103 24 81 57 36 78 36 116 85 Y. Onishchuk, H. Ono, Y. Onuki, P. Oskin, H. Ozaki, , P. Pakhlov, , G. Pakhlova, , A. Paladino, , T. Pang,109 A. Panta,107 E. Paoloni,89,40 S. Pardi,37 H. Park,53 S.-H. Park,21 B. Paschen,97 A. Passeri,42 A. Pathak,103 S P t 24 S P l 81 T K P dl 57 I P i 36 R P t t ik 78 M Pi l 36 L E Piil 116 P L M P d t L 85 T. Podobnik,78 S. Pokharel,107 G. Polat,1 V. Popov,23 C. Praz,10 S. Prell,49 E. Prencipe,15 M. T. Prim,97 N. Rad,10 P. Rados,10 S. Raiz,92,44 M. Remnev,3,68 I. Ripp-Baudot,95 M. Ritter,56 G. Rizzo,89,40 L. B. Rizzuto,78 S. H. Robertson,60,33 D. Rodríguez P´erez,85 J. M. Roney,115,33 A. Rostomyan,10 N. Rout,27 G. Russo,86,37 D. Sahoo,80 D. A. Sanders,107 S. Sandilya,26 A. Sangal,99 L. Santelj,102,78 Y. Sato,21 V. Savinov,109 B. Scavino,46 J. Search for B + →K + ν¯ν Decays Using an Inclusive Tagging Method at Belle II Behera,27 J. V. Bennett,107 E. Bernieri,42 F. U. Bernlochner,97 M. Bertemes,30 E. Bertholet,82 M. Bessner,101 S. Bettarini,89,40 F. Bianchi,91,43 T. Bilka,6 D. Biswas,103 A. Bozek,66 M. Bračko,105,78 P. Branchini,42 N. Braun,48 T. E. Browder,101 A. Budano,42 S. Bussino,90,42 M. Campajola,86,37 L. Cao,97 G. Casarosa,89,40 C. Cecchi,88,39 D. Červenkov,6 P. Chang,65 R. Cheaib,10 V. Chekelian,59 C. Chen,49 Y.-T. Chen,65 B. G. Cheon,20 K. Chilikin,54 K. Chirapatpimol,7 K. Cho,51 S.-J. Cho,120 S. Choudhury,26 D. Cinabro,117 L. Corona,89,40 L. M. Cremaldi,107 S. Cunliffe,10 T. Czank,114 F. Dattola,10 E. De La Cruz-Burelo,5 G. de Marino,94 G. De Nardo,86,37 M. De Nuccio,10 G. De Pietro,42 R. de Sangro,36 M. Destefanis,91,43 S. Dey,82 A. De Yta-Hernandez,5 A. Di Canto,2 F. Di Capua,86,37 J. Dingfelder,97 Z. Doležal,6 I. Domínguez Jim´enez,85 T. V. Dong,12 K. Dort,47 S. Dubey,101 S. Duell,97 G. Dujany,95 S. Eidelman,3,54,68 M. Eliachevitch,97 D. Epifanov,3,68 T. Ferber,10 D. Ferlewicz,106 T. Fillinger,95 G. Finocchiaro,36 S. Fiore,41 A. Fodor,60 F. Forti,89,40 A. Frey,17 B. G. Fulsom,71 N. Gabyshev,3,68 E. Ganiev,92,44 M. Garcia-Hernandez,5 A. Garmash,3,68 V. Gaur,116 A. Gaz,87,38 A. Gellrich,10 R. Giordano,86,37 A. Giri,26 A. Glazov,10 B. Gobbo,44 R. Godang,111 P. Goldenzweig,48 B. Golob,102,78 P. Grace,96 W. Gradl,46 E. Graziani,42 D. Greenwald,81 Y. Guan,99 K. Gudkova,3,68 C. Hadjivasiliou,71 S. Halder,80 K. Hara,21,19 O. Hartbrich,101 K. Hayasaka,67 H. Hayashii,64 S. Hazra,80 C. Hearty,98,33 I. Heredia de la Cruz,5,9 M. Hernández Villanueva,107 A. Hershenhorn,98 T. Higuchi,114 E. C. Hill,98 H. Hirata,62 M. Hoek,46 M. Hohmann,106 C.-L. Hsu,112 T. Humair,59 T. Iijima,62,63 K. Inami,62 G. Inguglia,30 J. Irakkathil Jabbar,48 A. Ishikawa,21,19 R. Itoh,21,19 M. Iwasaki,69 Y. Iwasaki,21 P. Jackson,96 W. W. Jacobs,28 D. E. Jaffe,2 Y. Jin,44 C. Joo,114 H. Junkerkalefeld,97 A. B. Kaliyar,80 J. Kandra,6 K. H. Kang,53 R. Karl,10 G. Karyan,119 T. Kawasaki,50 C. Ketter,101 H. Kichimi,21 C. Kiesling,59 C.-H. Kim,20 D. Y. Kim,77 Y.-K. Kim,120 T. D. Kimmel,116 P. Kodyš,6 T. Koga,21 S. Kohani,101 T. Konno,50 A. Korobov,3,68 S. Korpar,105,78 E. Kovalenko,3,68 T. M. G. Kraetzschmar,59 F. Krinner,59 P. Križan,102,78 P. Krokovny,3,68 T. Kuhr,56 , , , , y , g , , , , S. Korpar,105,78 E. Kovalenko,3,68 T. M. G. Kraetzschmar,59 F. Krinner,59 P. Križan,102,78 P. Krokovny,3,68 T. Kuhr,56 J. u a , . u a , . u a , . u a a, . u go, S. u , . u , . J. wo , S. acap a a, Y.-T. Lai,114 C. La Licata,114 L. Lanceri,44 J. S. Lange,47 M. Laurenza,90,42 K. (Belle II Collaboration) (Belle II Collaboration) 1Aix Marseille Universit´e, CNRS/IN2P3, CPPM, 13288 Marseille 2Brookhaven National Laboratory, Upton, New York 11973 3Budker Institute of Nuclear Physics SB RAS, Novosibirsk 630090 4Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 5Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional, Mexico City 07360 6Faculty of Mathematics and Physics, Charles University, 121 16 Prague 7Chiang Mai University, Chiang Mai 50202, Thailand 8Chiba University, Chiba 263-8522 9Consejo Nacional de Ciencia y Tecnología, Mexico City 03940 10Deutsches Elektronen–Synchrotron, 22607 Hamburg 11Duke University, Durham, North Carolina 27708 12Institute of Theoretical and Applied Research (ITAR), Duy Tan University, Hanoi 100000 13ENEA Casaccia, I-00123 Roma 14Earthquake Research Institute, University of Tokyo, Tokyo 113-0032 15Forschungszentrum Jülich, 52425 Jülich 16Key Laboratory of Nuclear Physics and Ion-beam Application (MOE) and Institute of Modern Physics, Fudan University, Shanghai 200443 17II. 181802-1 PHYSICAL REVIEW LETTERS 127, 181802 (2021) 8Gifu University, Gifu 501-1193 38INFN Sezione di Padova, I-35131 Padova 39 39INFN Sezione di Perugia, I-06123 Perugia 40 40INFN Sezione di Pisa, I-56127 Pisa 41 41INFN Sezione di Roma, I-00185 Roma 42 42INFN Sezione di Roma Tre, I-00146 Roma 43 43INFN Sezione di Torino, I-10125 Torino 44 44INFN Sezione di Trieste, I-34127 Trieste 45Advanced Science Research Center, Japan Atomic Energy Agency, Naka 319-1195 46 46Johannes Gutenberg-Universität Mainz, Institut für Kernphysik, D-55099 Mainz 47 47Justus-Liebig-Universität Gießen, 35392 Gießen mentelle Teilchenphysik, Karlsruher Institut für Technologie, 76131 Karlsruhe 49 48Institut für Experimentelle Teilchenphysik, Karlsruher Institut für Technologie, 76131 Karlsruhe 49I S U i i A I 50011 49Iowa State University, Ames, Iowa 50011 50 50Kitasato University, Sagamihara 252-0373 51Korea Institute of Science and Technology Information, Daejeon 34141 52 53Kyungpook National University, Daegu 41566 v Physical Institute of the Russian Academy of Sciences, Moscow 119991 55 54P. N. Lebedev Physical Institute of the Russian Academy of Sciences, Moscow 119991 55 l l 116029 56Ludwig Maximilians University, 80539 Munich (Belle II Collaboration) Physikalisches Institut, Georg-August-Universität Göttingen, 37073 Göttingen 18Gifu University, Gifu 501-1193 19The Graduate University for Advanced Studies (SOKENDAI), Hayama 240-0193 20Department of Physics and Institute of Natural Sciences, Hanyang University, Seoul 04763 21High Energy Accelerator Research Organization (KEK), Tsukuba 305-0801 22J-PARC Branch, KEK Theory Center, High Energy Accelerator Research Organization (KEK), Tsukuba 305-0801 23National Research University Higher School of Economics, Moscow 101000 24Indian Institute of Science Education and Research Mohali, SAS Nagar, 140306 25Indian Institute of Technology Bhubaneswar, Satya Nagar 751007 26Indian Institute of Technology Hyderabad, Telangana 502285 27Indian Institute of Technology Madras, Chennai 600036 28Indiana University, Bloomington, Indiana 47408 29Institute for High Energy Physics, Protvino 142281 30Institute of High Energy Physics, Vienna 1050 31Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8530 32Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049 33Institute of Particle Physics (Canada), Victoria, British Columbia V8W 2Y2 34Institute of Physics, Vietnam Academy of Science and Technology (VAST), Hanoi 35Instituto de Fisica Corpuscular, Paterna 46980 36INFN Laboratori Nazionali di Frascati, I-00044 Frascati 37INFN Sezione di Napoli, I-80126 Napoli 38INFN Sezione di Padova, I-35131 Padova 39INFN Sezione di Perugia, I-06123 Perugia 40INFN Sezione di Pisa, I-56127 Pisa 41INFN Sezione di Roma, I-00185 Roma 42INFN Sezione di Roma Tre, I-00146 Roma 43INFN Sezione di Torino, I-10125 Torino 44INFN Sezione di Trieste, I-34127 Trieste 45Advanced Science Research Center, Japan Atomic Energy Agency, Naka 319-1195 46Johannes Gutenberg-Universität Mainz, Institut für Kernphysik, D-55099 Mainz 47Justus-Liebig-Universität Gießen, 35392 Gießen 48Institut für Experimentelle Teilchenphysik, Karlsruher Institut für Technologie, 76131 Karlsruhe 49Iowa State University, Ames, Iowa 50011 50Kitasato University, Sagamihara 252-0373 51Korea Institute of Science and Technology Information, Daejeon 34141 52Korea University, Seoul 02841 53Kyungpook National University, Daegu 41566 54P. N. Lebedev Physical Institute of the Russian Academy of Sciences, Moscow 119991 55Liaoning Normal University, Dalian 116029 56Ludwig Maximilians University, 80539 Munich 181802 2 arch Institute, University of Tokyo, Tokyo 113-0032 PHYSICAL REVIEW LETTERS 127, 181802 (2021) 57Luther College, Decorah, Iowa 52101 58Malaviya National Institute of Technology Jaipur, Jaipur 302017 59Max-Planck-Institut für Physik, 80805 München 60McGill University, Montr´eal, Qu´ebec, H3A 2T8 61Moscow Physical Engineering Institute, Moscow 115409 62Graduate School of Science, Nagoya University, Nagoya 464-8602 63Kobayashi-Maskawa Institute, Nagoya University, Nagoya 464-8602 64Nara Women’s University, Nara 630-8506 65Department of Physics, National Taiwan University, Taipei 10617 66H. Niewodniczanski Institute of Nuclear Physics, Krakow 31-342 67Niigata University, Niigata 950-2181 68Novosibirsk State University, Novosibirsk 630090 69Osaka City University, Osaka 558-8585 70Research Center for Nuclear Physics, Osaka University, Osaka 567-0047 71Pacific Northwest National Laboratory, Richland, Washington 99352 72Panjab University, Chandigarh 160014 73Punjab Agricultural University, Ludhiana 141004 74Meson Science Laboratory, Cluster for Pioneering Research, RIKEN, Saitama 351-0198 75Showa Pharmaceutical University, Tokyo 194-8543 76Soochow University, Suzhou 215006 77Soongsil University, Seoul 06978 78J. 181802-2 181802-2 PHYSICAL REVIEW LETTERS 127, 181802 (2021) Stefan Institute, 1000 Ljubljana 79Taras Shevchenko National University of Kiev, Kiev 80Tata Institute of Fundamental Research, Mumbai 400005 81Department of Physics, Technische Universität München, 85748 Garching 82School of Physics and Astronomy, Tel Aviv University, Tel Aviv, 69978 83Toho University, Funabashi 274-8510 84Tokyo Institute of Technology, Tokyo 152-8550 85Universidad Autonoma de Sinaloa, Sinaloa 80000 86Dipartimento di Scienze Fisiche, Universit`a di Napoli Federico II, I-80126 Napoli 87Dipartimento di Fisica e Astronomia, Universit`a di Padova, I-35131 Padova 88Dipartimento di Fisica, Universit`a di Perugia, I-06123 Perugia 89Dipartimento di Fisica, Universit`a di Pisa, I-56127 Pisa 90Dipartimento di Matematica e Fisica, Universit`a di Roma Tre, I-00146 Roma 91Dipartimento di Fisica, Universit`a di Torino, I-10125 Torino 92Dipartimento di Fisica, Universit`a di Trieste, I-34127 Trieste 93Universit´e de Montr´eal, Physique des Particules, Montr´eal, Qu´ebec, H3C 3J7 94Universit´e Paris-Saclay, CNRS/IN2P3, IJCLab, 91405 Orsay 95Universit´e de Strasbourg, CNRS, IPHC, UMR 7178, 67037 Strasbourg 96Department of Physics, University of Adelaide, Adelaide, South Australia 5005 97University of Bonn, 53115 Bonn 98University of British Columbia, Vancouver, British Columbia, V6T 1Z1 99University of Cincinnati, Cincinnati, Ohio 45221 100University of Florida, Gainesville, Florida 32611 101University of Hawaii, Honolulu, Hawaii 96822 102Faculty of Mathematics and Physics, University of Ljubljana, 1000 Ljubljana 103University of Louisville, Louisville, Kentucky 40292 104National Centre for Particle Physics, University Malaya, 50603 Kuala Lumpur 105Faculty of Chemistry and Chemical Engineering, University of Maribor, 2000 Maribor 106School of Physics, University of Melbourne, Victoria 3010 107University of Mississippi, University, Mississippi 38677 108University of Miyazaki, Miyazaki 889-2192 109University of Pittsburgh, Pittsburgh, Pennsylvania 15260 110University of Science and Technology of China, Hefei 230026 111University of South Alabama, Mobile, Alabama 36688 112School of Physics, University of Sydney, New South Wales 2006 113Department of Physics, University of Tokyo, Tokyo 113-0033 Institute for the Physics and Mathematics of the Universe (WPI), University of Tokyo, Kashiwa 277-85 115U i i f Vi i Vi i B i i h C l bi V8W 3P6 96Department of Physics, University of Adelaide, Adelaide, South Australia 5005 97 97University of Bonn, 53115 Bonn 98University of British Columbia, Vancouver, British Columbia, V6T 1Z1 99 99University of Cincinnati, Cincinnati, Ohio 45221 100 0University of Florida, Gainesville, Florida 32611 101 101University of Hawaii, Honolulu, Hawaii 96822 102Faculty of Mathematics and Physics, University of Ljubljana, 1000 Ljubljana 103 103University of Louisville, Louisville, Kentucky 40292 104National Centre for Particle Physics, University Malaya, 50603 Kuala Lumpur 5 05Faculty of Chemistry and Chemical Engineering, University of Maribor, 2000 Maribor 106 ool of Physics, University of Melbourne, Victoria 3010 107University of Mississippi, University, Mississippi 38677 108 108University of Miyazaki, Miyazaki 889-2192 108University of Miyazaki, Miyazaki 889-2192 University of Pittsburgh, Pittsburgh, Pennsylvania 1 109University of Pittsburgh, Pittsburgh, Pennsylvania 15260 10 110University of Science and Technology of China, Hefei 230026 111 111University of South Alabama, Mobile, Alabama 36688 iversity of South Alabama, Mobile, Alabama 36688 113Department of Physics, University of Tokyo, Tokyo 113-0033 113Department of Physics, University of Tokyo, Tokyo 113-0033 114 te for the Physics and Mathematics of the Universe (WPI), University of Tokyo, Kashiwa 277-8583 115 115University of Victoria, Victoria, British Columbia, V8W 3P6 116Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 181802-3 Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Funded by SCOAP3. 181802-3 181802-3 DOI: 10.1103/PhysRevLett.127.181802 Flavor-changing neutral-current transitions, such as b →sν¯ν, are suppressed in the standard model (SM) by the extended Glashow-Iliopoulos-Maiani mechanism [1]. These transitions can only occur at higher orders in SM perturbation theory via weak amplitudes involving the exchange of at least two gauge bosons, as illustrated in Fig. 1. The absence of charged leptons in the final state reduces the theoretical uncertainty compared to similar b → sll transitions, which are affected by the breakdown of factorization due to photon exchange [2]. The branching fraction of the Bþ →Kþν¯ν decay [3], which involves a b →sν¯ν transition, is predicted to be ð4.6 0.5Þ × 10−6, where the main contribution to the uncertainty arises from the Bþ →Kþ transition form factor [4]. in a semileptonic decay [18,19]. This tagging suppresses background events but results in a low signal reconstruction efficiency, typically well below 1%. In all analyses reported to date, no evidence for a signal is found, and the current experimental upper limit on the branching fraction is estimated to be 1.6 × 10−5 at 90% confidence level [20]. in a semileptonic decay [18,19]. This tagging suppresses background events but results in a low signal reconstruction efficiency, typically well below 1%. In all analyses reported to date, no evidence for a signal is found, and the current experimental upper limit on the branching fraction is estimated to be 1.6 × 10−5 at 90% confidence level [20]. In this search, a novel and independent inclusive tagging approach is used, inspired by Ref. [21]. This approach has the benefit of a larger signal efficiency of about 4%, at the cost of higher background levels. The method exploits the distinctive topological and kinematic features of the Bþ → Kþν¯ν decay that distinguish this process from the seven dominant background categories. These are other decays of charged B mesons, decays of neutral B mesons, and the five continuum categories eþe−→q¯q with q ¼ u, d, s, c quarks and eþe−→τþτ−. The signal candidates are reconstructed as a single charged-particle trajectory (track) generated by the kaon, typically carrying higher momen- tum than background particles. The remaining tracks and energy deposits, referred to as the “rest of the event” (ROE), can thus be associated with the decay of the accompanying B meson. Furthermore, the neutrinos pro- duced in the signal B meson decay typically carry a significant fraction of its energy. PHYSICAL REVIEW LETTERS 127, 181802 (2021) PHYSICAL REVIEW LETTERS 127, 181802 (2021) (Received 6 May 2021; revised 23 July 2021; accepted 10 September 2021; published 27 October 2021) (Received 6 May 2021; revised 23 July 2021; accepted 10 September 2021; published 27 O A search for the flavor-changing neutral-current decay Bþ →Kþν¯ν is performed at the Belle II experiment at the SuperKEKB asymmetric energy electron-positron collider. The data sample corresponds to an integrated luminosity of 63 fb−1 collected at the ϒð4SÞ resonance and a sample of 9 fb−1 collected at an energy 60 MeV below the resonance. Because the measurable decay signature involves only a single charged kaon, a novel measurement approach is used that exploits not only the properties of the Bþ → Kþν¯ν decay, but also the inclusive properties of the other B meson in the ϒð4SÞ →B ¯B event, to suppress the background from other B meson decays and light-quark pair production. This inclusive tagging approach offers a higher signal efficiency compared to previous searches. No significant signal is observed. An upper limit on the branching fraction of Bþ →Kþν¯ν of 4.1 × 10−5 is set at the 90% confidence level. DOI: 10.1103/PhysRevLett.127.181802 The charged particle with the highest transverse momen- tum in each event, reconstructed with at least one hit in the PXD, is chosen to be the signal kaon candidate. Studies on simulated signal events show that the chosen candidate is the signal kaon in 78% of the cases. Furthermore, the signal candidate is required to satisfy PID requirements that suppress pion background. The PID requirements retain 62% of kaons while removing 97% of pions for events from the signal region, which is defined below. Simulated events are weighted to correct the dependence of the efficiency of this selection on the transverse momentum and the polar angle of the signal candidate, according to the efficiency observed in data. The remaining charged particles are fit to a common vertex and are attributed, together with the photons, to the ROE. A full description of the Belle II detector is given in Ref. [33]. The detector consists of several subdetectors arranged in a cylindrical structure around the beam pipe. Compared to its predecessor Belle [34], a pixel detector (PXD) has been added at a minimum radius of 1.4 cm. This improves the resolution of the impact parameter to about 12 μm in the transverse direction for high-momentum tracks [35], which helps to reject background events for this analysis. The PXD is surrounded by a four-layer double-sided silicon strip detector, referred to as the silicon vertex detector, and a central drift chamber (CDC). A time- of-propagation counter and an aerogel ring-imaging Cherenkov counter cover the barrel and forward end cap regions of the detector, respectively, and are essential for charged-particle identification (PID). The electromagnetic calorimeter (ECL) makes up the remaining volume inside a superconducting solenoid, which operates at 1.5 T. A dedicated detector to identify K0 L mesons and muons is installed in the outermost part of the detector. The z axis of the laboratory frame is defined as the symmetry axis of the solenoid, and the positive direction is approximately given by the incoming electron beam. The polar angle θ, as well as the longitudinal and the transverse direction are defined with respect to the z axis. The relevant online event- selection systems (triggers) for this analysis are based either on the number of tracks in the CDC or on the energy deposits in the calorimeter. DOI: 10.1103/PhysRevLett.127.181802 The specific properties of signal events are captured in a variety of discriminating variables used as inputs for event classifiers to separate signal from background. have longitudinal and transverse impact parameters with respect to the average interaction point of jdzj < 3 and dr < 0.5 cm, respectively. Photons are identified as energy deposits in the ECL exceeding 0.1 GeV that are within the CDC acceptance and are not matched to tracks. Each of the charged particles and photons is required to have an energy of less than 5.5 GeV to reject misreconstructed objects and cosmic muons. have longitudinal and transverse impact parameters with respect to the average interaction point of jdzj < 3 and dr < 0.5 cm, respectively. Photons are identified as energy deposits in the ECL exceeding 0.1 GeV that are within the CDC acceptance and are not matched to tracks. Each of the charged particles and photons is required to have an energy of less than 5.5 GeV to reject misreconstructed objects and cosmic muons. This search uses data from eþe−collisions produced in 2019 and 2020 by the SuperKEKB collider [22]. The data, corresponding to an integrated luminosity of 63 fb−1 [23], were recorded by the Belle II detector at a center-of-mass energy of ﬃﬃﬃs p ¼ 10.58 GeV, which corresponds to the ϒð4SÞ resonance, and contain 68 million B ¯B pairs [24]. An additional off-resonance sample of 9 fb−1 integrated luminosity, collected at an energy 60 MeV lower than the ϒð4SÞ resonance, is used to constrain the yields of continuum events. Events are required to contain no more than ten recon- structed tracks to suppress background events with only a small loss of signal efficiency. Low track-multiplicity background events are suppressed by demanding at least four tracks in the event. To further suppress such back- ground with a negligible loss of signal events, the total energy from all reconstructed objects in the event must exceed 4 GeV and the polar angle θ of the missing momentum must be between 17° and 160°. The signal and background samples are generated using a variety of event generators, summarized in the Supplemental Material [25], with the detector response simulated by the Belle II Analysis Software Framework (BASF2) [31], interfaced with GEANT4 [32]. The simulated Bþ →Kþν¯ν events are weighted according to the SM form-factor calculations from Ref. [2]. DOI: 10.1103/PhysRevLett.127.181802 The resulting “missing ” i d fi d h d d l In this search, a novel and independent inclusive tagging approach is used, inspired by Ref. [21]. This approach has the benefit of a larger signal efficiency of about 4%, at the cost of higher background levels. The method exploits the distinctive topological and kinematic features of the Bþ → Kþν¯ν decay that distinguish this process from the seven dominant background categories. These are other decays of charged B mesons, decays of neutral B mesons, and the five continuum categories eþe−→q¯q with q ¼ u, d, s, c quarks and eþe−→τþτ−. The signal candidates are reconstructed as a single charged-particle trajectory (track) generated by the kaon, typically carrying higher momen- tum than background particles. The remaining tracks and energy deposits, referred to as the “rest of the event” (ROE), can thus be associated with the decay of the accompanying B meson. Furthermore, the neutrinos pro- duced in the signal B meson decay typically carry a significant fraction of its energy. The resulting “missing momentum” is defined as the momentum needed to cancel Studies of this rare decay are currently of particular interest, as this process offers a complementary probe of potential non-SM physics scenarios that are proposed to explain the tensions with the SM predictions in b →slþl− transitions [5] observed in Refs. [6–11]. More generally, measurements of the Bþ →Kþν¯ν decay help constrain models that predict new particles, such as leptoquarks [12], axions [13], or dark matter particles [14]. The study of the Bþ →Kþν¯ν decay is experimentally challenging as the final state contains two neutrinos, which leave no signature in the detector and cannot be used to derive information about the signal B meson. Previous searches used tagged approaches, where the second B meson produced in the eþe−→ϒð4SÞ →B ¯B event is explicitly reconstructed in a hadronic decay [15–17] or FIG. 1. The lowest-order quark-level diagrams for the b →sν¯ν transition in the SM are either of the penguin (a) or the box (b) type. FIG. 1. The lowest-order quark-level diagrams for the b →sν¯ν transition in the SM are either of the penguin (a) or the box (b) type. 181802-4 PHYSICAL REVIEW LETTERS 127, 181802 (2021) the sum of the three-momenta of all reconstructed tracks and energy deposits in the center-of-mass system of the incoming beams. DOI: 10.1103/PhysRevLett.127.181802 p Simulated signal and background events are used to train binary event classifiers, which are based on the FastBDT algorithm [37], a multivariate method that uses boosted decision trees (BDTs). Several inputs are considered for this process, including general event-shape variables described in Ref. [38], as well as variables characterizing the kaon-candidate and the kinematic properties of the ROE. Moreover, vertices of two and three charged par- ticles, including the kaon candidate, are reconstructed to identify potential kaons from D0 and Dþ meson decays, and variables describing the fit quality and kinematic properties of the vertices are derived. Variables that are not well described by the simulation and those that do not contribute to the separation power of the classification are removed. This results in a set of 51 training variables, summarized in the Supplemental Material [25]. A first binary classifier BDT1 is trained on approxi- mately 106 simulated events of each of the seven consid- ered background categories and on the same number of signal events. The most discriminating variables are found to be event-shape variables, specifically the reduced Fox- Wolfram moment R1, which measures the momentum imbalance in the event where the signal tends to be The events are reconstructed using BASF2. The trajecto- ries of charged particles are determined using the algorithms described in Ref. [36]. Charged particles are required to have a transverse momentum pT > 0.1 GeV=c, to be within the CDC acceptance (17° < θ < 150°), and to 181802-5 PHYSICAL REVIEW LETTERS 127, 181802 (2021) output of 0.9 < BDT1 < 0.99 and BDT2 < 0.7 in the ϒð4SÞ on-resonance data and corresponding simulation. The study confirms the accurate modeling of the BDT distributions by the simulation for a sample of events that have similar kinematic properties as signal events, while containing only a negligible contribution from signal. imbalanced due to signal neutrinos [39], and the modified Fox-Wolfram variables that are functions of the missing momentum and of the momentum of the signal kaon candidate [40]. To improve the training performance at high BDT1 values, a second classifier BDT2 is trained with the same set of input variables as BDT1 on events with BDT1 > 0.9, which corresponds to a signal efficiency of 28% and a purity of 0.02%. DOI: 10.1103/PhysRevLett.127.181802 The training is performed using a simulated background sample of 100 fb−1 equivalent luminosity (corresponding to a total of 5 × 106 events with BDT1 > 0.9) and a sample of 1.5 × 106 signal events with BDT1 > 0.9. An increase of 35% in signal purity is achieved by the additional application of BDT2 on top of BDT1, when comparing the performance at a signal efficiency of 4%. BDT1 and BDT2 use the same set of FastBDT parameters [37], which are optimized based on a grid search in the parameter space and are specified in the Supplemental Material [25]. g y g g g The decay Bþ →KþJ=ψ with J=ψ →μþμ−is used as an independent validation channel, exploiting its large branching fraction and distinctive experimental signature. These events are selected in data and Bþ →KþJ=ψ simulation by requiring the presence of two muons with an invariant mass within 50 MeV=c2 of the known J=ψ mass [20]. To suppress background events, the variable jΔEj ¼ jE B − ﬃﬃﬃs p =2j is required to be less than 100 MeV and the beam-energy constrained mass Mbc ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ s=ð4c4Þ −p2 B =c2 p is required to exceed 5.25 GeV=c2, where E B and p B are given by the energy and the magnitude of the three-momentum of the signal B meson candidate defined in the center-of-mass system of the incoming beams. This results in 1720 events being selected in the data sample at an expected background contamina- tion of 5%. Each event is then reconsidered as a Bþ → Kþν¯ν event by ignoring the muons from the J=ψ decay and replacing the momentum of the kaon candidate with the generator-level momentum of the kaon in a randomly selected Bþ →Kþν¯ν event from simulation. The same modifications are applied to the data and Bþ →KþJ=ψ simulation. The results are summarized in Fig. 2, where the distributions of the output values of both BDTs are shown. Good agreement between simulation and data is observed for the selected events before (Bþ →KþJ=ψ→μþμ−) and An additional binary classifier is used to correct for mismodeling of continuum simulation, following a data- driven method presented in Ref. [41]. More information about the implementation is included in the Supplemental Material [25]. A comparison of simulated continuum events with off-resonance data shows that the application of the derived event weight improves the modeling of all input variables. DOI: 10.1103/PhysRevLett.127.181802 A signal region (SR) is defined to be BDT1 > 0.9 and BDT2 > 0.95 and is further divided into 3 × 3 bins in the BDT2 × pTðKþÞ space, where pTðKþÞ is the transverse momentum of the kaon candidate. The bin boundaries, decided by minimizing the expected upper limit on the signal branching fraction, are [0.95, 0.97, 0.99, 1.0] in BDT2 and ½0.5; 2.0; 2.4; 3.5 GeV=c in pTðKþÞ. Furthermore, three control regions are used to help con- strain the background yields. The control region CR1 consists of 1 × 3 bins in the BDT2 × pTðKþÞ space, defined at lower values of BDT2 ∈½0.93; 0.95 and using the same pTðKþÞ bins as the SR. The two other control regions, CR2 and CR3, consist of off-resonance data with identical BDT2 and pTðKþÞ ranges and bins as in the SR and CR1, respectively. FIG. 2. Distribution of the classifier output BDT1 (main figure) and BDT2 for BDT1 > 0.9 (inset). The distributions are shown before (J=ψ→μþμ−) and after (J=ψ→= μþ= μ−) the muon removal and update of the kaon-candidate momentum of selected Bþ →KþJ=ψ events in simulation (MC) and data. As a reference, the classifier outputs directly obtained from simulated Bþ →Kþν¯ν signal events are overlaid. The simulation histograms are scaled to the total number of Bþ →KþJ=ψ events selected in data. The expected yields of the SM signal and the back- grounds in the SR are 14 and 844 events, respectively, corresponding to a signal efficiency of 4.3%. In most of these background events, a Kþ produced in a D meson decay is selected as the signal kaon candidate. To enable the study of other, non-SM signal models, the fraction of signal events in the SR is studied as a function of the generated dineutrino invariant mass squared q2. The efficiency is 13% for q2 ¼ 0 and drops to zero for q2 > 16 GeV2=c4. The full distribution can be found in the Supplemental Material [25]. FIG. 2. Distribution of the classifier output BDT1 (main figure) and BDT2 for BDT1 > 0.9 (inset). The distributions are shown before (J=ψ→μþμ−) and after (J=ψ→= μþ= μ−) the muon removal and update of the kaon-candidate momentum of selected Bþ →KþJ=ψ events in simulation (MC) and data. As a reference, the classifier outputs directly obtained from simulated Bþ →Kþν¯ν signal events are overlaid. PHYSICAL REVIEW LETTERS 127, 181802 (2021) Shifts of the nuisance parameters corresponding to the seven background sources are investigated before μ is revealed. The parameters corresponding to the continuum background yields are increased by, at most, one standard deviation, which confirms that they are not pulled sub- stantially in the fit given the observed difference in the normalization of the continuum simulation with respect to the off-resonance data. The background yields in the bins of CR2 and CR3 predicted by the fit are found in agreement with the off-resonance data. No shift is observed for the parameters corresponding to the background yields from charged and neutral B meson decays, which are the dominant contributions in the most sensitive SR bins. after (Bþ →KþJ=ψ→= μþ= μ−) the modifications. The two- sample Kolmogorov-Smirnov p values [42] for the BDT1 and BDT2 distributions of simulation and data, after the modifications, are 7% and 23%, respectively. The ratio of the selection efficiencies BDT1 > 0.9, BDT2 > 0.95 in data and simulation is found to be 1.06 0.10. The statistical analysis to determine the signal yields is performed with the PYHF package [43,44], which constructs a binned likelihood following the HISTFACTORY [45] formalism. The templates for the yields of the signal and the seven background categories are derived from simu- lation. The likelihood function is a product of Poisson probability density functions that combine the information from all 24 signal- and control-region bins defined on the on- and off-resonance data. The systematic uncertainties discussed below are included in the likelihood as nuisance parameters that are event-count modifiers with correspond- ing constraints modeled as normal probability density functions. The parameter of interest, the signal strength μ, is defined as a factor relative to the SM expectation and is determined simultaneously with the nuisance parameters using a simultaneous maximum-likelihood fit to the binned distribution of data event counts. A comparison of the data and fit results in the SR and CR1 is shown in Fig. 3. The corresponding figure for CR2 and CR3 can be found in the Supplemental Material [25]. The signal purity is found to be 6% in the SR and is as high as 22% in the three bins with BDT2 > 0.99. Continuum events make up 59% of the background in the SR and 28% of the events with BDT2 > 0.99. DOI: 10.1103/PhysRevLett.127.181802 The simulation histograms are scaled to the total number of Bþ →KþJ=ψ events selected in data. The performance of the classifiers BDT1 and BDT2 on data is tested by selecting events with a moderate BDT 181802-6 181802-6 PHYSICAL REVIEW LETTERS 127, 181802 (2021) PHYSICAL REVIEW LETTERS 127, 181802 (2021) The signal strength is determined by the fit to be μ ¼ 4.2þ3.4 −3.2 ¼ 4.2þ2.9 −2.8ðstatÞþ1.8 −1.6ðsystÞ, where the statistical uncertainty is estimated using pseudo-experiments based on Poisson statistics. The total uncertainty is obtained by a profile likelihood scan, fitting the model with fixed values of μ around the best-fit value, while keeping the other fit parameters free. The systematic uncertainty is calcu- lated by subtracting the statistical uncertainty in quadrature from the total uncertainty. An additional 10% theoretical uncertainty arising from the knowledge of the branching The leading systematic uncertainty is the normalization uncertainty on the background yields. The yields of the seven individual background categories are allowed to float independently in the fit. However, each of them is con- strained assuming a normal constraint, centered at the expected background yield obtained from simulation and a standard deviation corresponding to 50% of the central value. This value is motivated by a global normalization difference of ð40 12Þ% between the off-resonance data and simulation in the control regions CR2 and CR3 and also covers the uncertainty on the sample luminosity. The remaining considered systematic uncertainties may also influence the shape of the templates. Systematic uncertain- ties originating from the branching fractions of the leading B meson decays, the PID correction, and the SM form factors are accounted for with three nuisance parameters each to model correlations between the individual SR and CR bins. The remaining systematic uncertainties arise from the energy miscalibration of hadronic and beam-back- ground calorimeter energy deposits and the tracking inef- ficiency, and are each accounted for with one nuisance parameter. The systematic uncertainty due to the limited size of simulated samples is taken into account by one nuisance parameter per bin per background category. This results in a total of 175 nuisance parameters. FIG. 3. Yields in on-resonance data and as predicted by the simultaneous fit to the on- and off-resonance data, corresponding to an integrated luminosity of 63 and 9 fb−1, respectively. The predicted yields are shown individually for charged and neutral B meson decays and the sum of the five continuum categories. The leftmost three bins belong to CR1 with BDT2 ∈½0.93; 0.95 and the other nine bins correspond to the SR, three for each range of BDT2 ∈½0.95; 0.97; 0.99; 1.0. Each set of three bins is defined by pTðKþÞ ∈½0.5; 2.0; 2.4; 3.5 GeV=c. PHYSICAL REVIEW LETTERS 127, 181802 (2021) 26400255, the National Institute of Informatics, and Science Information NETwork 5 (SINET5), and the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan; National Research Foundation (NRF) of Korea Grants No. 2016R1D1A1B01010135, No. 2016R1D1A1B02012900, No. 2018R1A2B3003643, No.2018R1A6A1A06024970,No.2018R1D1A1B07047294, N 2019K1A3A7A09033840 dN 2019R1I1A3A01058933 ratio in the SM is not included. The result corresponds to a branching fraction of the Bþ →Kþν¯ν decay of ½1.9þ1.6 −1.5 × 10−5 ¼ ½1.9þ1.3 −1.3ðstatÞþ0.8 −0.7ðsystÞ × 10−5. 1.5 1.3 0.7 This value is statistically compatible with the measure- ments performed by previous experiments. Details are given in the Supplemental Material [25]. The uncertainty on the branching fraction is used to define a measure to compare the performance of the individual tagging tech- niques. Assuming that this uncertainty scales as the inverse square root of the integrated luminosity [46], the inclusive approach is more than a factor of 3.5 better per integrated luminosity than the hadronic tagging of Ref. [16], approx- imately 20% better than the semileptonic tagging of Ref. [19], and approximately 10% better than the combined hadronic and semileptonic tagging of Ref. [17]. Moreover, the events in the SR differ from the ones selected by the hadronic and semileptonic tagging so that a statistical combination of the measurements provides additional sensitivity. The inclusive tagging approach can be applied to a variety of decay modes, such as Bþ →Kþν¯ν and Bþ →τþν, and, because of the increased signal efficiency, this can be done on smaller data samples than required for semileptonic or hadronic tagging. As no significant signal is observed, the expected and observed upper limits on the branching fraction are determined using the CLs method [47], a modified fre- quentist approach that is based on a profile likelihood ratio [48]. The expected 90% confidence level (C. L.) upper limit on the Bþ →Kþν¯ν branching fraction of 2.3 × 10−5 is derived assuming a background-only hypothesis. The observed upper limit is 4.1 × 10−5 at the 90% C.L. The full distribution of the determined CLs values is shown in the Supplemental Material [25]. In summary, a search for the rare decay Bþ →Kþν¯ν is performed using an inclusive tagging approach, which has not previously been used to study this process. This analysis uses data corresponding to an integrated luminos- ity of 63 fb−1 collected at the ϒð4SÞ resonance by the Belle II detector, as well as an off-resonance sample correspond- ing to 9 fb−1. PHYSICAL REVIEW LETTERS 127, 181802 (2021) All yields in the right- most three bins are scaled by a factor of 2. To validate the fitting software, an alternative approach based on a simplified Gaussian likelihood function (SGHF) is developed. Tests of both PYHF and SGHF are performed using pseudo-experiments, in which both statistical and systematic uncertainties are taken into account, including background normalizations. No bias in μ and its uncertainty is observed, and the p value for the data and fit model compatibility is found to be above 65%. 181802-7 PHYSICAL REVIEW LETTERS 127, 181802 (2021) Australian Research Council and research Grants No. DP180102629, No. DP170102389, No. DP170102204, No. DP150103061, No. FT130100303, No. FT130100018, and No. FT120100745; Austrian Federal Ministry of Education, Science and Research, Austrian Science Fund No. P 31361-N36, and Horizon 2020 ERC Starting Grant No. 947006 “InterLeptons”; Natural Sciences and Engineering Research Council of Canada, Compute Canada, and CANARIE; Chinese Academy of Sciences and research Grant No. QYZDJ-SSW-SLH011, National Natural Science Foundation of China and research Grants No. 11521505, No. 11575017, No. 11675166, No. 11761141009, No. 11705209, and No. 11975076, LiaoNing Revitalization Talents Program under Contract No. XLYC1807135, Shanghai Municipal Science and Technology Committee under Contract No. 19ZR1403000, Shanghai Pujiang Program under Grant No. 18PJ1401000, and the CAS Center for Excellence in Particle Physics (CCEPP); the Ministry of Education, Youth, and Sports of the Czech Republic under Contract No. LTT17020 and Charles University Grants No. SVV 260448 and No. GAUK 404316; European Research Council, Seventh Framework PIEF-GA-2013-622527, Horizon 2020 ERC-Advanced Grants No. 267104 and No. 884719, Horizon 2020 ERC- Consolidator Grant No. 819127, Horizon 2020 Marie Sklodowska-Curie Grant Agreement No. 700525 “NIOBE,” and Horizon 2020 Marie Sklodowska-Curie RISE project JENNIFER2 Grant Agreement No. 822070 (European grants); L’Institut National de Physique Nucl´eaire et de Physique des Particules (IN2P3) du CNRS (France); BMBF, DFG, HGF, MPG, and AvH Foundation (Germany); Department of Atomic Energy under Project Identification No. RTI 4002 and Department of Science and Technology (India); Israel Science Foundation Grant No. 2476/17, U.S.-Israel Binational Science Foundation Grant No. 2016113, and Israel Ministry of Science Grant No. 3-16543; Istituto Nazionale di Fisica Nucleare and the research grants BELLE2; Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research Grants No. 16H03968, No. 16H03993, No. 16H06492, No. 16K05323, No. 17H01133, No. 17H05405, No. 18K03621, No. 18H03710, No. 18H05226, No. 19H00682, No. 26220706, and No. PHYSICAL REVIEW LETTERS 127, 181802 (2021) Abudin´en et al. 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Korea Institute of Science and Technology Information, and KREONET/GLORIAD; Universiti Malaya RU grant, Akademi Sains Malaysia, and Ministry of Education Malaysia; Frontiers of Science Program Contracts No. FOINS-296, No. CB-221329, No. CB-236394, No. CB-254409, and No. CB-180023, and SEP- CINVESTAV research Grant No. 237 (Mexico); the Polish Ministry of Science and Higher Education and the National Science Center; the Ministry of Science and Higher Education of the Russian Federation, Agreement No. 14.W03.31.0026, and the HSE University Basic Research Program, Moscow; University of Tabuk research Grants No. S-0256-1438 and No. S-0280-1439 (Saudi Arabia); Slovenian Research Agency and research Grants No. J1-9124 and No. P1-0135; Agencia Estatal de Investigacion, Spain Grants No. FPA2014-55613-P and No. FPA2017-84445-P, and CIDEGENT/2018/020 of Generalitat Valenciana; Ministry of Science and Technology and research Grants No. MOST106-2112-M- 002-005-MY3 and No. MOST107-2119-M-002-035-MY3, and the Ministry of Education (Taiwan); Thailand Center of Excellence in Physics; TUBITAK ULAKBIM (Turkey); Ministry of Education and Science of Ukraine; the U.S. National Science Foundation and research Grants No. PHY- 1807007 and No. PHY-1913789, and the U.S. Department of Energy and research Awards No. DE-AC06-76RLO1830, No. DE-SC0007983, No. DE-SC0009824, No. DE- SC0009973, No. DE-SC0010073, No. DE-SC0010118, No. DE-SC0010504, No. DE-SC0011784, No. DE- SC0012704, No. DE-SC0021274; and the Vietnam Academy of Science and Technology (VAST) under Grant No. DL0000.05/21-23. 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PHYSICAL REVIEW LETTERS 127, 181802 (2021) No statistically significant signal is observed and an upper limit on the branching fraction of 4.1 × 10−5 at the 90% C.L. is set, assuming a SM signal. This measurement is competitive with previous results for similar integrated luminosities, demonstrating the capabil- ity of the inclusive tagging approach, which is widely applicable and expands the future physics reach of Belle II. We thank the SuperKEKB group for the excellent operation of the accelerator; the KEK cryogenics group for the efficient operation of the solenoid; the KEK computer group for on-site computing support; and the raw-data centers at BNL, DESY, GridKa, IN2P3, and INFN for off-site computing support. This work was sup- ported by the following funding sources: Science Committee of the Republic of Armenia Grant No. 20TTCG-1C010; 181802-8 PHYSICAL REVIEW LETTERS 127, 181802 (2021) PHYSICAL REVIEW LETTERS 127, 181802 (2021) Aaij et al. (LHCb Collaboration), J. 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B149, 413 (1979); B157, 543(E) (1979). [46] The product σBR ﬃﬃﬃﬃ L p of the total branching-fraction uncer- tainty σBR and the integrated luminosity of the data sample L is used as a measure, assuming that σBR scales as 1= ﬃﬃﬃﬃ L p . [40] S. H. Lee et al. (Belle Collaboration), Phys. Rev. Lett. 91, 261801 (2003). [41] D. Martschei, M. Feindt, S. Honc, and J. Wagner-Kuhr, J. Phys. Conf. Ser. 368, 012028 (2012). [47] A. L. Read, J. Phys. G 28, 2693 (2002). [48] G. Cowan, K. Cranmer, E. Gross, and O. Vitells, Eur. Phys. J. C 71, 1554 (2011); 73, 2501(E) (2013). [42] J. L. Hodges, Arkiv för Matematik 3, 469 (1958). 181802-10
https://openalex.org/W2046898658	https://europepmc.org/articles/pmc3753321?pdf=render	English	null	Ex-Vivo Dynamic 3-D Culture of Human Tissues in the RCCS™ Bioreactor Allows the Study of Multiple Myeloma Biology and Response to Therapy	PloS one	2,013	cc-by	9,457	Abstract Three-dimensional (3-D) culture models are emerging as invaluable tools in tumor biology, since they reproduce tissue- specific structural features and cell-cell interactions more accurately than conventional 2-D cultures. Multiple Myeloma, which depends on myeloma cell-Bone Marrow microenvironment interactions for development and response to drugs, may particularly benefit from such an approach. An innovative 3-D dynamic culture model based on the use of the RCCSTM Bioreactor was developed to allow long-term culture of myeloma tissue explants. This model was first validated with normal and pathological explants, then applied to tissues from myeloma patients. In all cases, histological examination demonstrated maintenance of viable myeloma cells inside their native microenvironment, with an overall well preserved histo-architecture including bone lamellae and vessels. This system was then successfully applied to evaluate the cytotoxic effects exerted by the proteasome inhibitor Bortezomib not only on myeloma cells but also on angiogenic vessels. Moreover, as surrogate markers of specialized functions expressed by myeloma cells and microenvironment, b2 microglobulin, VEGF and Angiopoietin-2 levels, as well as Matrix Metalloproteases activity, were evaluated in supernatants from 3D cultures and their levels reflected the effects of Bortezomib treatment. Notably, determination of b2 microglobulin levels in supernatants from Bortezomib-treated samples and in patients’sera following Bortezomib-based therapies disclosed an overall concordance in the response to the drug ex vivo and in vivo. Our findings indicate, as a proof of principle, that 3-D, RCCSTM bioreactor-based culture of tissue explants can be exploited for studying myeloma biology and for a pre-clinical approach to patient-targeted therapy. Citation: Ferrarini M, Steimberg N, Ponzoni M, Belloni D, Berenzi A, et al. (2013) Ex-Vivo Dynamic 3-D Culture of Human Tissues in the RCCSTM Bioreactor Allows the Study of Multiple Myeloma Biology and Response to Therapy. PLoS ONE 8(8): e71613. doi:10.1371/journal.pone.0071613 Editor: Venugopalan Cheriyath, Texas A&M University, United States of America Received March 8, 2013; Accepted July 1, 2013; Published August 26, 2013 Copyright: 2013 Ferrarini 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: 2013 Ferrarini 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. Abstract Funding: This work was supported by AIRC-Special Program Molecular Clinical Oncology AIRC 561000 project Nu 9965 (to FCC), by European Union Grant LSHB- CT-2006-037168 (EXERA) to GM and by research funding from Bosh S.p.A. to EF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have the following interests. This study was partly funded by Bosh S.p.A. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. he authors have the following interests. This study was partly funded by Bosh S.p.A. There are no patents, products in development o clare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the * E-mail: ferrarini.marina@hsr.it (MF); ferrero.elisabetta@hsr.it (EF) * E-mail: ferrarini.marina@hsr.it (MF); ferrero.elisabetta@hsr.it (EF) . These authors contributed equally to this work. . These authors contributed equally to this work. . These authors contributed equally to this work. " These authors also contributed equally to this work. " These authors also contributed equally to this work. Ex-Vivo Dynamic 3-D Culture of Human Tissues in the RCCSTM Bioreactor Allows the Study of Multiple Myeloma Biology and Response to Therapy Marina Ferrarini1., Nathalie Steimberg2., Maurilio Ponzoni3, Daniela Belloni1, Angiola Berenzi4, Stefania Girlanda5, Federico Caligaris-Cappio1,6, Giovanna Mazzoleni2", Elisabetta Ferrero1" 1 Department of Oncology, San Raffaele Scientific Institute, Milan, Italy, 2 Laboratory of Tissue Engineering, Department of Clinical and Experimental Sciences, School of Medicine, University of Brescia, Brescia, Italy, 3 Pathology Unit, San Raffaele Scientific Institute, Milan, Italy, 4 Institute of Pathological Anatomy, Department of Clinical and Experimental Sciences, School of Medicine, University of Brescia, Brescia, Italy, 5 Bone Marrow Transplantation Unit, San Raffaele Scientific Institute, Milan, Italy, 6 Vita- Salute San Raffaele University School of Medicine, Milan, Italy Citation: Ferrarini M, Steimberg N, Ponzoni M, Belloni D, Berenzi A, et al. (2013) Ex-Vivo Dynamic 3-D Culture of Human Tissues in the RCCSTM Bioreactor Allows the Study of Multiple Myeloma Biology and Response to Therapy. PLoS ONE 8(8): e71613. doi:10.1371/journal.pone.0071613 Bioreactor-Based 3-D Culture of Myeloma Explants Bioreactor-Based 3-D Culture of Myeloma Explants architecture. This is especially true for the vascular compartment, that critically contributes to MM progression, thus representing a major target for anti-MM therapies [15–18]. As an example, the proteasome inhibitor Bortezomib is considered to be an effective anti-MM drug, not only for its cytotoxic effect against MM cells, but also for its anti-angiogenic activity [19–22]. Culture of tissue explants in static condition is an alternative 3-D system, but the reduced mass transport (gas/nutritional support and waste removal) achieved [23] limits its application to morpho-functional analyses. carcinoma (RCC) samples were obtained from patients undergo- ing tumor resection. Written informed consent was obtained from patients (or from the parents in the case of the minor), in accordance with the Declaration of Helsinki and the approval for use of primary samples was obtained from the Institutional Review Board of the San Raffaele Scientific Institute. Patient 1 was a forty-year-old man, newly diagnosed with MM. Patient 2 was a 66-year-old man with a 3-year history of micromolecular MM, who developed new osteolytic lesions requiring vertebroplasty. Patient 3 was a 75-year-old man with a not-secretory myeloma evolved from plasmocytoma. These three patients did not receive any therapy at the time when vertebroplasty was performed. To circumvent these limitations and to provide new models that more closely resemble the in vivo situation in patients, we applied a 3-D dynamic culture system, based on the use of the innovative microgravity technology provided by the Rotary Cell Culture System (RCCSTM) Bioreactor, to ex-vivo culture of MM specimens. In this dynamic, horizontally rotating bioreactor, the culture vessel is fluid-filled, so that no headspace is left between atmosphere and culture medium. All during the experimental procedures, RCCSTM operational conditions were constantly monitored and optimized for obtaining a laminar flow of the fluid medium inside the culture chamber, while the 3D tissue samples were maintained suspended in a relatively stable position (condition of ‘‘free fall’’), so that shear forces and turbulence normally associated with impeller-driven stirred bioreactors were reduced to a minimum. These hydrodynamic conditions related to the culture vessel rotation allow also to offset tissue sedimentation. Finally, the peculiar structure of the vessel (that includes a gas exchange membrane) favours the optimal oxygenation of thick, 3D samples, reducing the occurrence of the central core necrosis often observed with other bioreactors. Bioreactor-Based 3-D Culture of Myeloma Explants The main advantages of our culture model, in comparison to conventional 2-D and 3-D static culture systems, are presented in the Figure S1. Patient 4 was a 61-year-old woman who developed a subcutaneous frontal mass infiltrating the right orbit. Histological examination of the lesion showed neoplastic PC monoclonal for l light chain. She was treated with thalidomide plus dexamethasone and autologous hematopoietic stem cell transplantation (HSCT) and she died after two weeks of pneumonitis and septic shock. We obtained tissue biopsies from skull bone at the diagnosis, before any medical treatment. Patient 5 was a 62-year-old man with a 3-year history of micromolecular MM. Despite different lines of therapy, including Bortezomib-based therapy, autologous HSCT and allogeneic HSCT from matched unrelated donor (MUD), the disease progressed and the patient developed subcutaneous abdominal localizations, which were biopsied for diagnosis and submitted to culture in Bioreactor. Histology showed infiltration with mono- clonal PC expressing l light chain. A salvage therapy with Bortezomib, liposomal doxorubicin and dexamethasone was started, but the patient developed invasive aspergillosis and Staphylococcus epidermidis sepsis and died nine months after allogeneic HSCT. By providing an optimal balance between increased mass transfer and deleterious effects of shear stress, this dynamic bioreactor, as already demonstrated by our group, generates the best conditions for long-term in vitro maintenance of cell viability and function in 3-D cell culture systems, multicellular engineered tissue-like constructs, and tissue explants [9,24]. Thus, our strategy was to capitalize the advantages offered by the RCCSTM bioreactor to the culture of MM tissues, and particularly of human BM samples, whose complexity is not fully reproduced by currently available models. Baseline characteristics of MM patients are summarized in Table 1. Tissue Cultures in RCCSTM Bioreactor 3-D, dynamic culture of biopsies was performed using the RCCSTM bioreactor RCCS-1 (Synthecon Inc., Houston TX, USA), in 10 ml-HARV culture vessels, equipped with a gas exchange membrane made of silicon rubber, that allows an optimal diffusion of oxygen inside the vessel [24]. Bioreactor was kept inside an incubator, with humidified atmosphere at 37uC and 95% air 5% CO2. During the experimental procedures, RCCSTM operational conditions, settled in order to keep samples in a ‘‘free fall’’ condition that maximise mass transfer, were constantly monitored, in order to reduce to a minimum the mechanical stress (shear forces) on the explants surface and to prevent samples sedimentation. Normal skin and bone, as well as RCC and MM explants (2–3 mm3 of maximal volume) were cultured for three up to 14 days in RPMI plus 10% Fetal Calf Serum (tissue culture medium, TCM). To evaluate the effects of Bortezomib (PS-341, VelcadeH, Millenium Pharmaceuti- cals, Cambridge MA) on MM tissue, the drug was added at 50 nM to parallel cultures. Tissue explants were harvested at 3–4 days Animals Young (7–8 week-old) male Sprague-Dawley rats (Harlan- Europe, Milan, Italy) whose body weight ranged from 200 to 225 g were used as bone donors, in accordance with Italian National Guidelines for the care and use of laboratory animals. Tibial bones were collected, processed and cut as described [24]. Studies on animal models were approved by the Ethical Committee of the San Raffaele Scientific Institute and carried out according to the prescribed guidelines. The RCCSTM Bioreactor was firstly evaluated for its ability to preserve skin architecture and blood vessel integrity. The system was further validated for its capacity to maintain the complex bone/BM microenvironment with whole rat tibia epiphyses, and also bone explants from healthy donors. We finally applied the same strategy to tissue explants from MM patients, including BM and extra-medullary localization. Herein we demonstrate, as a proof of principle, that our dynamic, 3-D culture model allows to explore, for extended time periods, morphological and functional features of MM tissue components (vessels included) as well as their specific sensitivity to drug(s). Introduction traditionally relied on two-dimensional (2-D) cell culture models [8]. Indeed, recent studies documented differences between cell morphology and behaviour in static, 2D formats compared with more physiological 3D microenvironments [7,9,10]. Accordingly, 3-D approaches, which better reproduce in vivo-like responses, have become a focus of intense investigation [11–13]. In this context, a major breakthrough in the study of MM was achieved by Kirshner et al. [14], who proposed a new 3-D culture model, which enables clonal expansion of MM PC as well as testing of anti-tumor drugs. Multiple Myeloma (MM) is a plasma cell (PC) tumor. Tight interactions between MM cells and Bone Marrow (BM) microen- vironment are central to MM pathogenesis as they allow the delivery of pro-survival signals and confer chemo-resistance to neoplastic cells [1–3]. Indeed, tumor microenvironment is emerging as a major determinant in tumor initiation, progression and response to therapy in both haematological [4] and solid tumors [5,6]. Hence, the generation of in vitro models able to recapitulate tumor microenvironment is critical for both under- standing cancer biology and assessing its vulnerability to drugs [7]. While in vivo tissues and organs are three- dimensional (3-D), our capacity to understand their formation, function, and pathology Currently available 3-D systems include ex vivo models, based on the use of isolated cells and tissue-derived components (tissue engineering methods). However, these models do not fully reproduce the complexity and specificity of the native tissue August 2013 \| Volume 8 \| Issue 8 \| e71613 1 PLOS ONE \| www.plosone.org August 2013 \| Volume 8 \| Issue 8 \| e71613 Histochemistry and Immunohistochemistry Serial 5 mm thick sections were stained with hematoxylin and eosin (H&E) and with anti-CD34 mAb (QBEnd/10, Novocastra, Newcastle upon Tyne, UK), as previously described [25]. Anti- Podoplanin, Clone D2-40 (DAKO) was used to identify lymphatic vessels. Microvascular density (MVD) quantification was per- formed as described [26]. Patients Tissue explants were obtained from five MM patients and included BM biopsies obtained at vertebroplasty for patient 1, 2 and 3, skull bone from patient 4, and a skin lesion from patient 5. BM biospsies from three elderly patients undergoing hip replacement (HD1, HD2, HD3) and a 14-year-old boy undergoing knee arthroplasty were used as controls. Skin samples were obtained from patients undergoing abdominal surgery. Renal cell August 2013 \| Volume 8 \| Issue 8 \| e71613 PLOS ONE \| www.plosone.org 2 Bioreactor-Based 3-D Culture of Myeloma Explants Table 1. Demographic and clinical characteristics of MM patients. Patient Age/sex Diagnosis DSS ISS Therapies before biopsy Tissue Pt1 40/M MM IgGl IIIA 3 None BM Pt 2 66/M MM mmoll IIA 3 Btz+Doxo; ARA-C, Btz+Mel+auto-HSCT BM Pt 3 75/M MM nsl IIIA 3 Mel, Prednisone, Thal BM Pt 4 61/F MM IgG l IIIA 3 None Skull Pt 5 62/M MM mmol l IIIA nk Thal/Dex+double auto-HSCT; Btz/Dex+Mel+third auto-HSCT; Btz/Dex/Doxo+MUD transpl. Skin Abbreviations: Thal. thalidomide; Btz, Bortezomib; Dex, Dexamethasone; Doxo, Doxorubicin; Mel, Melphalan; nk, not known; ns, not secretory; mmol, micromolecular. doi:10.1371/journal.pone.0071613.t001 Abbreviations: Thal. thalidomide; Btz, Bortezomib; Dex, Dexamethasone; Doxo, Doxorubicin; Mel, Melphalan; nk, not known; ns, not secretory; mmol, micromolecular. doi:10.1371/journal.pone.0071613.t001 intervals, formalin-fixed, decalcified and paraffin-embedded. Con- comitantly, supernatants from culture chambers were withdrawn and frozen at 220uC until use, and fresh TCM was added. As a control, when indicated, parallel cultures of normal skin and bone explants as well as of RCC in static conditions were performed in Petri dishes and maintained under the same culture conditions as for cultures in Bioreactor. conditions, setting up parallel cultures of skin and RCC explants. As shown in Figure 1, skin cultured in the RCCSTM Bioreactor for up to one week completely preserved the overall architecture, in which all strata were distinguishable. Moreover, both blood and lymphatic (D2-40 staining) vessels could be identified by morpho- logical and immuno-histochemical analyses and preserved a patent lumen, surrounded by an intact, continuous lining of endothelial cells (EC) (Fig. 1 A, insert). Conversely, parallel cultures in static conditions exhibited consistent disruption and diffuse loss of integrity, which prevented the unequivocal identification of single strata and also vessels (Fig. 1A). RCC samples cultured in Bioreactor for up to seven days also exhibited preserved overall histoarchitecture and cell viability, at variance with those cultured under static conditions (Fig. 1A). Cell isolation and apoptosis PC were isolated from Patient 4’s skull lesion by density centrifugation [28]. PC death upon 24-hrs in vitro treatment with Bortezomib 50 nM was assessed by propidium iodide (PI) staining and FACS analysis [29] and by Transmission Electron Microscopy (TEM), as described [30]. Determination in supernatants of b2 microglobulin, VEGF, Angiopoietin-2, and of metalloproteases (MMP)-2 and -9 Determination in supernatants of b2 microglobulin, VEGF, Angiopoietin-2, and of metalloproteases (MMP)-2 and -9 p It is well known that MM develops almost exclusively inside BM, so far, however, suitable models which retain native human- BM microenvironment are not available. We therefore evaluated this culture model for its ability to maintain bone explants for long- term culture. To this aim, proximal epiphyses of rat tibia were cultured either in dynamic 3D conditions (RCCSTM Bioreactor), or in 2D static conditions. As for the skin model, dynamic conditions preserved histo-architecture and cell viability inside bone explants, whereas in static conditions, deprivation of cellular component was progressively observed. As a result, cells sponta- neously outgrown from tibia epiphyses began to proliferate to form a monolayer and they completely lost their native microenviron- ment as well as their normal organisation and morphology (Figure S2). Suitability of RCCSTM Bioreactor also for 3-D culture of MM explants, was then assessed by 3D culturing human bone biopsies from healthy donors. We analyzed samples from both elderly donors, age-matched with MM patients, and from a 14-year-old donor. Tissue explants (2–3 mm3 in maximal volume) were kept in 3-D dynamic culture for up to two weeks; at intervals of 3–4 days, specimens were retrieved and submitted to histopathological examination. As shown in Figure 1B, H&E staining identified intact BM architecture, consistent with age-related features, which was maintained throughout the culture period. In particular, normal bone explants, obtained from elderly subjects, displayed the classical, physiological, age-related prevalence of adipose tissue (Figure 1B, upper panel). Conversely, in BM explants from the young donor, nucleated, heterogeneous cellular elements were diffusely distributed in the context of abundant stroma (Figure 1B, middle panel). These morphological features were maintained throughout culture for up to 14 days (Figure S3A). Supernatants from untreated or Bortezomib-treated MM explants and bone controls were serially retrieved and frozen. Samples were subjected to SDS-PAGE in 10% polyacrylamide gel, containing 1 mg/ml gelatine (Sigma), to assess MMP-2 and MMP-9 activities as described [27]. VEGF and Angiopoietin-2 (Ang-2) concentrations were determined by ELISA. b2 micro- globulin concentration was determined by particle enhanced immunonephelometry. Statistical analysis Correlation between b2 microglobulin, VEGF and Ang-2 levels in supernatants from five MM patients (either treated or untreated with Bortezomib) was assessed using the Spearman’s rho coefficient (r). A p-value of #0.05 was considered statistically significant. RCCSTM Bioreactor preserves tissue architecture and blood vessels integrity BM from the elderly patient is physiologically depleted of hematopoietic components, and replaced by adipose tissue. Bone trabeculae are also evident (upper panels). BM from a young donor displays a substantial cellularity, represented by hematopoietic elements and adipocytes in the context of abundant stroma (middle panels). In parallel, bone explants from the young donor, cultured in conventional static conditions, display progressive loss of both cellular and stromal components, resulting in disruption of overall architecture (lower panels). Original Magnification (OM) 200X. doi:10.1371/journal.pone.0071613.g001 RCCSTM Bioreactor preserves tissue architecture and blood vessels integrity We first validated the actual advantage of culturing tissue explants in Bioreactor over standard tissue culture in static August 2013 \| Volume 8 \| Issue 8 \| e71613 PLOS ONE \| www.plosone.org 3 Bioreactor-Based 3-D Culture of Myeloma Explants Figure 1. 3-D culture in the RCCSTM bioreactor preserves cellularity and tissue histo-architecture. A. Left: skin explants from healthy donors were cultured in the RCCSTM bioreactor (upper panels) and in static conditions (lower panels) for seven days. Epidermal and dermal strata and annexes are well preserved and vessels, both blood (insert, arrows) and lymphatic (D2-40) are identifiable in dynamic conditions (upper panels), while histo-architecture is completely disrupted in static conditions (lower panels). Right: RCC samples cultured for seven days in Bioreactor (upper panels) showed maintenance of cellularity and viability (insert); both features were lost in parallel cultures under static conditions (lower panels). B. Bone explants from an elderly (upper panels) and a 14-year-old (middle panels) healthy control were cultured in Biorector; samples were retrieved at the indicated time points and submitted to H&E staining. BM from the elderly patient is physiologically depleted of hematopoietic components, and replaced by adipose tissue. Bone trabeculae are also evident (upper panels). BM from a young donor displays a substantial cellularity, represented by hematopoietic elements and adipocytes in the context of abundant stroma (middle panels). In parallel, bone explants from the young donor, cultured in conventional static conditions, display progressive loss of both cellular and stromal components, resulting in disruption of overall architecture (lower panels). Original Magnification (OM) 200X. doi:10.1371/journal.pone.0071613.g001 Figure 1. 3-D culture in the RCCSTM bioreactor preserves cellularity and tissue histo-architecture. A. Left: skin explants from healthy donors were cultured in the RCCSTM bioreactor (upper panels) and in static conditions (lower panels) for seven days. Epidermal and dermal strata and annexes are well preserved and vessels, both blood (insert, arrows) and lymphatic (D2-40) are identifiable in dynamic conditions (upper panels), while histo-architecture is completely disrupted in static conditions (lower panels). Right: RCC samples cultured for seven days in Bioreactor (upper panels) showed maintenance of cellularity and viability (insert); both features were lost in parallel cultures under static conditions (lower panels). B. Bone explants from an elderly (upper panels) and a 14-year-old (middle panels) healthy control were cultured in Biorector; samples were retrieved at the indicated time points and submitted to H&E staining. August 2013 \| Volume 8 \| Issue 8 \| e71613 3-D culture in Bioreactor of MM explants allows the assessment of their sensitivity to Bortezomib To further substantiate the reliability of our culture system in discriminating the impact of drugs on MM cells and on different microenvironmental players as well, we considered samples obtained from two patients with divergent clinical history and response to therapy, i.e., the Bortezomib-sensitive Patient 4 and the Bortezomib-resistant Patient 5 (Table 1). In particular, in Patient 4, a newly diagnosed MM, tissue was obtained from a skull lesion; a part of it was kept in 3-D culture, while another portion was used to isolate single PC suspension. Isolated PC resulted sensitive to Bortezomib in vitro, as indicated by PI incorporation at FACS Our culture system was further exploited to evaluate the effects of Bortezomib treatment on MM tissue explants. To this purpose, MM specimens from vertebroplasty were cultured in Bioreactor in the presence of Bortezomib for three days, in accordance with the kinetics of anti-myeloma activity exerted by the drug in vitro [31]. Indeed, Bortezomib severely affected PC viability, as assessed by morphological criteria, including cell size reduction, acidophilic cytoplasmic stain, nuclear pyknosis, karyolysis and karyorrhexis (Figure 2A, inserts). Accordingly, MM cells isolated from the BM Figure 2. Culture in Bioreactor allows the identification of single cell components inside MM microenvironment and of their individual response to Bortezomib. MM samples from a vertebral lesion (Patient 1) and from extramedullary localizations of the Bortezomib- sensitive Patient 4 and of the Bortezomib-resistant Patient 5 were cultured in the RCCSTM Bioreactor for up to seven days in the presence (+)/ absence(2) of Bortezomib 50 nM. H&E staining (Panel A and B) demonstrates the persistence of MM cells in untreated samples and the differential sensitivity to Bortezomib treatment. OM 2006for Patient 1, 1006for Patient 4 and 400 for Patient 5. Inserts are zoom of the corresponding pictures. Immunohistochemistry with anti-CD34 mAb Panel C) allows to identify vasculature and its vulnerability to Bortezomib (OM 2006). In Panel D, MVD was quantified at day 0 and after 7 days in the presence and absence of Bortezomib upon staining with CD34 mAb. doi:10.1371/journal.pone.0071613.g002 Figure 2. Culture in Bioreactor allows the identification of single cell components inside MM microenvironment and of their individual response to Bortezomib. Bioreactor-Based 3-D Culture of Myeloma Explants Bioreactor-Based 3-D Culture of Myeloma Explants were still evident in MM explants cultured in Bioreactor for 7 (Figure 2B) and 14 (Figure S3C) days. of Patients 1, 2 and 3 and cultured under static conditions appeared to be sensitive to Bortezomib treatment (50 nM) as indicated by the percentage of Annexin V+/PI+cells at FACS analysis (71%; 64% and 52%, respectively at 24 hrs, and almost 100% at three days in all cases). 3-D culture in Bioreactor of MM explants allows the assessment of their sensitivity to Bortezomib 3-D dynamic culture in the RCCSTM Bioreactor of MM tissues allows the maintenance and identification of individual compartments culturing tissue explants from BM, and offers a consistent advantage over presently available, conventional tissue culture systems. To validate this culture model for the assessment of MM microenvironment morphology as well as for the impact of drugs on its components, we utilized tissue explants from MM patients, including three bone lesions (from Patients 1, 2 and 3) and two extra-medullary lesions (from Patient 4 and Patient 5) (Table 1). Bone samples were obtained at vertebroplasty when patients were still untreated (Table 1). Histological analyses performed on BM explants cultured for three days in Bioreactor allowed the identification of single components of MM microenvironment, including MM cells, bone lamellae and vessels (arteriolae, arrows), within an overall well preserved architecture (Figure 2A). MM cells To validate the advantage of culture in the RCCSTM Bioreactor also for BM tissue, we set paralleled cultures of BM explants from a young donor in a conventional static condition. To this aim, we plated tissue explants of similar size in culture dishes in TCM under the same culture conditions adopted for culture in Bioreactor. When compared to matched samples from 3-D cultures carried out for up to two weeks (Figure 1B and Figure S3A), explants maintained in traditional cultures displayed progressive and striking disappearance of hematopoietic precur- sors and adipocytes, together with disruption of overall architec- ture (Figure 1B, lower panels and Figure S3B). Taken together, these data indicate that RCCSTM Bioreactor is suitable for August 2013 \| Volume 8 \| Issue 8 \| e71613 August 2013 \| Volume 8 \| Issue 8 \| e71613 PLOS ONE \| www.plosone.org 4 3-D culture in Bioreactor of MM explants allows the assessment of their sensitivity to Bortezomib MM samples from a vertebral lesion (Patient 1) and from extramedullary localizations of the Bortezomib- sensitive Patient 4 and of the Bortezomib-resistant Patient 5 were cultured in the RCCSTM Bioreactor for up to seven days in the presence (+)/ absence(2) of Bortezomib 50 nM. H&E staining (Panel A and B) demonstrates the persistence of MM cells in untreated samples and the differential sensitivity to Bortezomib treatment. OM 2006for Patient 1, 1006for Patient 4 and 400 for Patient 5. Inserts are zoom of the corresponding pictures. Immunohistochemistry with anti-CD34 mAb Panel C) allows to identify vasculature and its vulnerability to Bortezomib (OM 2006). In Panel D, MVD was quantified at day 0 and after 7 days in the presence and absence of Bortezomib upon staining with CD34 mAb. doi:10.1371/journal.pone.0071613.g002 August 2013 \| Volume 8 \| Issue 8 \| e71613 PLOS ONE \| www.plosone.org 5 Bioreactor-Based 3-D Culture of Myeloma Explants Figure 3. MM samples express specialized functions in culture. Supernatants from Patients’ explants, cultured for 3 days in the presence (grey columns) or absence (white columns) of Bortezomib (Btz, 50 nM), were retrieved and analyzed for b2 microglobulin (A), VEGF (B) and Ang-2 (C) concentrations. In A, B and C, b2 microglobulin, VEGF and Ang-2 concentrations in three days culture supernatants from five MM samples were compared to those from three healthy donors (HD). In D, correlation between b2 microglobulin, and VEGF and Ang-2 concentrations in Patients’ supernatants was assessed using the Spearman’s rho coefficient (r). (* p,0,05; *p,0.01.) doi:10.1371/journal.pone.0071613.g003 Bioreactor-Based 3-D Culture of Myeloma Explants Figure 3. MM samples express specialized functions in culture. Supernatants from Patients’ explants, cultured for 3 days in the presence (grey columns) or absence (white columns) of Bortezomib (Btz, 50 nM), were retrieved and analyzed for b2 microglobulin (A), VEGF (B) and Ang-2 (C) concentrations. In A, B and C, b2 microglobulin, VEGF and Ang-2 concentrations in three days culture supernatants from five MM samples were compared to those from three healthy donors (HD). In D, correlation between b2 microglobulin, and VEGF and Ang-2 concentrations in Patients’ supernatants was assessed using the Spearman’s rho coefficient (r) ( p,0 05; *p,0 01 ) Figure 3. MM samples express specialized functions in culture. 3-D culture in Bioreactor of MM explants allows the assessment of their sensitivity to Bortezomib Supernatants from Patients’ explants, cultured for 3 days in the presence (grey columns) or absence (white columns) of Bortezomib (Btz, 50 nM), were retrieved and analyzed for b2 microglobulin (A), VEGF (B) and Ang-2 (C) concentrations. In A, B and C, b2 microglobulin, VEGF and Ang-2 concentrations in three days culture supernatants from five MM samples were compared to those from three healthy donors (HD). In D, correlation between b2 microglobulin, and VEGF and Ang-2 concentrations in Patients’ supernatants was assessed using the Spearman’s rho coefficient (r). ( p,0,05; **p,0.01.) doi:10.1371/journal.pone.0071613.g003 MM samples cultured in the RCCSTM Bioreactor express metabolic, specialized functions that may be affected by Bortezomib treatment analysis and by nuclear condensation and intense cytoplasmic vacuolization at TEM (Figure S4). On the other hand, patient 5 had failed several lines of therapy and was under treatment with Bortezomib when he developed subcutaneous localizations, which were excised and submitted to 3-D culture in Bioreactor. Histological analyses performed on 3-D culture samples from the two patients allowed to identify PC (Figure 2B) and to assess their differential response to Bortezomib. In Patient 4, following a 7 days treatment with Bortezomib, PC disappeared and were replaced by stroma, in agreement with the findings observed when isolated PC were exposed to Bortezomib in vitro. Conversely, Bortezomib treatment did not affect MM PC in tissue explants retrieved from Patient 5, a feature that corroborated the inefficacy observed in vivo (Figure 2B). MM-associated microvasculature was highlighted by immuno-histochemical analysis with anti-CD34 mAb (Figure 2C). Notably, the impact of Bortezomib on microvasculature inside MM microenvironment was confirmed by decreased MVD in Bortezomib-treated MM tissue explants from both Patient 4 and Patient 5 (Figure 2D), irrespective of differential cytotoxicity on MM PC. analysis and by nuclear condensation and intense cytoplasmic vacuolization at TEM (Figure S4). On the other hand, patient 5 had failed several lines of therapy and was under treatment with Bortezomib when he developed subcutaneous localizations, which were excised and submitted to 3-D culture in Bioreactor. Histological analyses performed on 3-D culture samples from the two patients allowed to identify PC (Figure 2B) and to assess their differential response to Bortezomib. In Patient 4, following a 7 days treatment with Bortezomib, PC disappeared and were replaced by stroma, in agreement with the findings observed when isolated PC were exposed to Bortezomib in vitro. 3-D culture in Bioreactor of MM explants allows the assessment of their sensitivity to Bortezomib Conversely, Bortezomib treatment did not affect MM PC in tissue explants retrieved from Patient 5, a feature that corroborated the inefficacy observed in vivo (Figure 2B). MM-associated microvasculature was highlighted by immuno-histochemical analysis with anti-CD34 mAb (Figure 2C). Notably, the impact of Bortezomib on microvasculature inside MM microenvironment was confirmed by decreased MVD in Bortezomib-treated MM tissue explants from both Patient 4 and Patient 5 (Figure 2D), irrespective of differential cytotoxicity on MM PC. We then assessed whether also specialized functions expressed by MM cells and their microenvironment were maintained in 3-D culture in the RCCSTM bioreactor. To this aim, we first looked at b2 microglobulin concentration in culture supernatants from MM explants retrieved at different time intervals. Indeed, b2 micro- globulin serum levels significantly correlate with PC proliferation index [32] and are commonly used for staging of MM. As shown in Figure 3A, b2 microglobulin was detectable in supernatants obtained after three days of culture; moreover, b2 microglobulin levels decreased in the presence of Bortezomib in all samples, but in patient 5, in accordance with the drug sensitivity of MM cells shown by morphological examination. b2 microglobulin levels were instead negligible in supernatants from healthy bone explants of comparable size derived from 3 controls (HD) and cultured in the same experimental conditions (Figure 3A). Overproduction by MM cells of pro-angiogenic cytokines, including VEGF and Ang-2, is responsible for MM-induced angiogenic switch and in turn for MM progression [15–17,33]. Indeed, VEGF and Ang-2 could be detected in supernatants after PLOS ONE \| www.plosone.org August 2013 \| Volume 8 \| Issue 8 \| e71613 August 2013 \| Volume 8 \| Issue 8 \| e71613 6 Bioreactor-Based 3-D Culture of Myeloma Explants Figure 4. Kinetics of MMP-9 and MMP-2 activities and of VEGF secretion in culture supernatants. A: Supernatants from biopsies of 5 MM Patients and 3 healthy donors (HD), cultured for 3 days in the presence or absence of Bortezomib (Btz, 50 nM), were assessed for MMP-2 and MMP-9 activities. Zymography of both MMPs is shown (upper panels) and quantitated by densitometry (lower panels). White columns represent samples obtained in the absence of Bortezomib while grey columns in the presence of the drug. TC, black columns, represent negative controls. Kinetics of VEGF (B), pro-MMP and MMP activities (C) were determined in supernatants obtained from Patient 4’s and Patient 5’s samples. 3-D culture in Bioreactor of MM explants allows the assessment of their sensitivity to Bortezomib MM explants were kept in culture in bioreactor in the presence (grey) or absence (white) of Bortezomib (Btz, 50 nM); supernatants were retrieved at different time intervals and submitted to ELISA for VEGF determination (B) and to zymographic analysis (C) to assess MMP-9 and MMP-2 enzymatic activities. a.u. = arbitrary units. doi:10 1371/journal pone 0071613 g004 Figure 4. Kinetics of MMP-9 and MMP-2 activities and of VEGF secretion in culture supernatants. A: Supernatants from biopsies of 5 MM Patients and 3 healthy donors (HD), cultured for 3 days in the presence or absence of Bortezomib (Btz, 50 nM), were assessed for MMP-2 and MMP-9 activities. Zymography of both MMPs is shown (upper panels) and quantitated by densitometry (lower panels). White columns represent samples obtained in the absence of Bortezomib while grey columns in the presence of the drug. TC, black columns, represent negative controls. Kinetics of VEGF (B), pro-MMP and MMP activities (C) were determined in supernatants obtained from Patient 4’s and Patient 5’s samples. MM explants were kept in culture in bioreactor in the presence (grey) or absence (white) of Bortezomib (Btz, 50 nM); supernatants were retrieved at different time intervals and submitted to ELISA for VEGF determination (B) and to zymographic analysis (C) to assess MMP-9 and MMP-2 enzymatic activities. a.u. = arbitrary units. doi:10 1371/journal pone 0071613 g004 doi:10.1371/journal.pone.0071613.g004 activity, which was substantially lower than that from patient’s samples in the case of MMP-2, while did not differ from that of most patients in the case of MMP-9. three days of culture; moreover, their secretion was inhibited upon Bortezomib treatment with the exception of patient 5, closely paralleling the results obtained with b2 microglobulin release. Again, supernatants from controls contained very low levels of both VEGF and Ang-2 (Figure 3B–C). Notably, VEGF and Ang-2 expression positively and significantly correlated with that of b2 microglobulin (r = 0.679; p,0.05; r = 0.779; p,0.01, respectively) and also correlated with each other (r = 0.0890; p,0.01) (Figure 3D). Long-term maintenance of MM specialized functions in the RCCSTM Bioreactor To investigate whether culture in Bioreactor sustains proper differentiated functions over time, we took advantage of samples derived from the prototypical Patients 4 and 5. Supernatants obtained at different time points showed a progressive increase in VEGF concentration (Figure 4B). Notably, VEGF concentration abated in the presence of Bortezomib in the case of Patient 4, while it was unaffected in the case of Patient 5, once again paralleling the sensitivity to the drug of MM cells (Figure 4B). MMP-2, and to a lesser degree MMP-9 activity, also increased in supernatants from both patients for up to day 7, when a partial conversion of the proMMPs into their activated form can be observed. In Patient 4, Bortezomib treatment mildly affected both MM is characterized by accelerated production of the proteolytic enzymes MMP, which are critically involved in tumor growth, angiogenesis, homing and the development of osteolytic lesions, all typical events encountered in MM progression [34,35]. Zymography performed on day three supernatants from MM tissue explants showed MMP-2 and, to a lesser degree, MMP-9 activity. Both MMP-2 and MMP-9 activity varied among patients, as did the response to Bortezomib treatment (Figure 4A). Supernatants from controls also contained measurable MMP August 2013 \| Volume 8 \| Issue 8 \| e71613 PLOS ONE \| www.plosone.org August 2013 \| Volume 8 \| Issue 8 \| e71613 7 Bioreactor-Based 3-D Culture of Myeloma Explants Figure 5. b2 microglobulin levels in patients’sera and in supernatants from cultures in the RCCSTM Bioreactor. A: b2 microglobulin concentrations were determined in patients’sera before (white circles) and after (black circles) Bortezomib-based therapies (6axis). B: b2 microglobulin levels in 3 days supernatants from MM explants cultured in bioreactor in the absence (white) or presence (grey) of Bortezomib. Abbreviations: Bortezomib, Btz; Thal, thalidomide; Dex, Dexamethasone; Cy, Cyclophosphamide; Mel, Melphalan; HSCT, Hematopoietic Stem Cell Transplantation; Doxo, Doxorubicin. doi:10.1371/journal.pone.0071613.g005 Figure 5. b2 microglobulin levels in patients’sera and in supernatants from cultures in the RCCSTM Bioreactor. A: b2 microglobulin concentrations were determined in patients’sera before (white circles) and after (black circles) Bortezomib-based therapies (6axis). B: b2 microglobulin levels in 3 days supernatants from MM explants cultured in bioreactor in the absence (white) or presence (grey) of Bortezomib. Abbreviations: Bortezomib, Btz; Thal, thalidomide; Dex, Dexamethasone; Cy, Cyclophosphamide; Mel, Melphalan; HSCT, Hematopoietic Stem Cell Transplantation; Doxo, Doxorubicin. doi:10.1371/journal.pone.0071613.g005 pro and active MMPs, while in Patient 5 no effect could be determined (Figure 4C). Effect of Bortezomib on b2 microglobulin levels in patients’ sera and supernatants from cultures in Bioreactor The dynamic RCCSTM bioreactor-based tissue culture method, taking advantage on the unique milieu generated by simulated microgravity conditions [39,40], characterized by low shear and turbulence, by optimal O2 and nutrients delivery and waste removal [41], allows culture of MM explants, preserving the proper topographic and functional interactions between myeloma cells and the hosting microenvironment. In such conditions, tissue culture could be maintained for up to two weeks, thus substantially extending the duration (few days) achieved with currently available methods [38,39]. The model could be further implemented to recapitulate more closely physical features of the BM, and in particular hypoxia [42], whose impact on MM cell behaviour and response to drugs deserves to be investigated. Finally, to investigate the suitability of culture in Bioreactor to predict sensitivity to drugs, we compared b2 microglobulin levels in patients’ sera before and after Bortezomib-based therapies with those observed in supernatants from cultures in presence/absence of Bortezomib. In particular, we analyzed Patients 1, 2, 3 and 5; Patient 4 was not included, since she did not receive Bortezomib treatment (see Materials and Methods section). Figure 5 demon- strates an overall concordance between the effects exerted by Bortzomib in vivo and ex vivo. Long-term maintenance of MM specialized functions in the RCCSTM Bioreactor MMP-9 and -2 activities were also detectable in supernatants from HD samples; however, their levels did not increase over time and the active form could not be appreciated (Fig. S3 D). Currently available conventional static and dynamic culture systems (shacking or rolling platforms) fail to provide optimal gas/nutritional support (and waste removal) thus resulting in reduced viability of tissue explants and impaired cell morphology and function [37–38]. Accordingly, the comparison of normal (skin biopsies and BM explants) as well as cancer tissues cultured in static vs dynamic conditions provided by the RCCSTM Bioreactor clearly demonstrates the advantage of the latter, in terms of cellularity and histo-architecture. Supporting Information Figure S1 Major characteristics and advantages of dynamic culture in Bioreactor in comparison to the static one. Figure S1 Major characteristics and advantages of dynamic culture in Bioreactor in comparison to the static one. While several anti-myeloma agents, including Bortezomib, are endowed with anti-angiogenic properties, the actual effects on MM–associated vessels cannot be easily estimated [22]. Specifi- cally, neither imaging techniques nor reliable bio-markers of ongoing angiogenesis, which are being applied especially to solid tumors, have been validated so far for MM [22]. In the present RCCSTM-based model, the anti-angiogenic effect of Bortezomib inside MM microenvironment could be appreciated and measured by means of MVD quantification. Notably, we have previously shown that the cytotoxicity exerted by Bortezomib in vitro was directed against proliferating/activated EC, reminiscent of MM- associated EC [44], but not against quiescent EC [21]. While several anti-myeloma agents, including Bortezomib, are endowed with anti-angiogenic properties, the actual effects on MM–associated vessels cannot be easily estimated [22]. Specifi- cally, neither imaging techniques nor reliable bio-markers of ongoing angiogenesis, which are being applied especially to solid tumors, have been validated so far for MM [22]. In the present RCCSTM-based model, the anti-angiogenic effect of Bortezomib inside MM microenvironment could be appreciated and measured by means of MVD quantification. Notably, we have previously shown that the cytotoxicity exerted by Bortezomib in vitro was directed against proliferating/activated EC, reminiscent of MM- associated EC [44], but not against quiescent EC [21]. Figure S2 Dynamic culture in Bioreactor of rat tibial explants preserves histo-architecture. Tibial proximal epiphyses from young rats were submitted to dynamic, 3-D culture in RCCS Bioreactor for up to two weeks, retrieved at weekly intervals and stained with H&E. Evidence of hematopoietic elements and adipose tissue inside a well preserved BM architecture is assessable throughout the culture period, while in static conditions the majority of cells leave BM already at 7 days and progressively formed a classical 2-D monolayer culture. OM: 2006 for 3-D culture, 4006 for 2-D culture. (TIF) Figure S3 Long-term culture of normal BM and MM samples in Bioreactor. BM samples from a young donor were splitted in parallel dynamic (A) and static (B) cultures and maintained for 14 days. In C, MM sample from patient 5 was kept in culture for up to 2 weeks in Bioreactor. OM: 2006; insert is a zoom of the corresponding picture. Acknowledgments We thank Fabio Ciceri, Magda Marcatti, Moreno Tresoldi and Giovanni Tonon, Myeloma Unit (HSR-IRCCS), for precious advice and discussion and for providing specimens and clinical details. The authors are grateful to Giacomo Dell’Antonio (Pathology Unit, HSR) for his expertise on RCC specimens; to Jennifer Boniotti (Brescia University), Ilenia Papa and Arianna Vino (HSR) for skilled technical assistance during tissue explants preparation and culturing; and to Mauro Motta (LaboRaf, HSR) for b2 microglobulin determination. Bone explants were kindly provided by Giuseppe Gioia (Department of Orthopedics, HSR, Milan). 3. Hideshima T, Mitsiades C, Tonon G, Richardson PG, Anderson KC (2007). Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets. Nat Rev Cancer 7: 585–598. Discussion Also in this case, however, vessels are neo-formed inside engrafting BMSC. Supporting Information A: MM cells isolated from a skull lesion were treated for 24 hrs with Bortezomib (50 nM). CD138+ PC death was determined by FACS analysis by Propidium Iodide (PI) staining. B: TEM on MM cells treated with Bortezomib (50 nM) for 24 hrs shows nuclear condensation and massive cytoplasmic vacuolization, at variance with control (N.T.), that displays intact nuclei and cytoplasmic organelles. Discussion We herein demonstrate that some important biological aspects of MM, and response to drugs of individual cellular components inside MM microenvironment, can be reliably evaluated with a 3- D culture system in RCCSTM Bioreactor. Among all available 3-D culture methods, culture of normal and pathological tissues represents the most relevant, since it enables the ex vivo study of cellular processes, in a multicellular context in which the original tissue architecture and microenvironment are maintained [23,36]. Notably, histological analyses performed on serially retrieved MM explants allow the monitoring of the response to anti- myeloma drugs. This was particularly evident when samples from two prototypical Patients, were examined. Our 3-D culture system also offers the unique opportunity to assess the response to drugs of native MM associated vessels, whose structure and quantification over time can be only partially evaluated in conventional culture August 2013 \| Volume 8 \| Issue 8 \| e71613 August 2013 \| Volume 8 \| Issue 8 \| e71613 PLOS ONE \| www.plosone.org 8 Bioreactor-Based 3-D Culture of Myeloma Explants currently available models for the study of MM cells-BM interaction [48]; moreover, it can be further exploited for screening of new anti-myeloma drugs [49] and for a pre-clinical approach to patient-targeted therapy of MM. systems. Indeed, the 3-D model previously described by Kirshner et al. [14], based on the in vitro reconstruction of MM microenvironment, fails to recapitulate the original structure of the tissue vasculature. More recently, an additional 3-D model of human MM microenvironment has been developed, based upon the implantation into a SCID mouse of polymeric scaffolds coated by human BM stromal cells (BMSC) and then repopulated in vivo with human MM cells [43]. This model represents a further advance, especially for preclinical evaluation of anti-MM agents in a proper microenvironmental context. Also in this case, however, vessels are neo-formed inside engrafting BMSC. systems. Indeed, the 3-D model previously described by Kirshner et al. [14], based on the in vitro reconstruction of MM microenvironment, fails to recapitulate the original structure of the tissue vasculature. More recently, an additional 3-D model of human MM microenvironment has been developed, based upon the implantation into a SCID mouse of polymeric scaffolds coated by human BM stromal cells (BMSC) and then repopulated in vivo with human MM cells [43]. This model represents a further advance, especially for preclinical evaluation of anti-MM agents in a proper microenvironmental context. Author Contributions In conclusion, our findings indicate that the RCCSTM Bioreactor permits culture of MM explants allowing assessment of metabolic activity and drugs sensitivity of MM cells and their microenvironment, particularly vessels. This model complements Conceived and designed the experiments: MF MP FC-C GM EF. Performed the experiments: NS MP DB AB. Analyzed the data: MF MP GM EF. Wrote the paper: MF SG GM EF. Gathering of clinical data: SG. Conceived and designed the experiments: MF MP FC-C GM EF. Performed the experiments: NS MP DB AB. Analyzed the data: MF MP GM EF. Wrote the paper: MF SG GM EF. Gathering of clinical data: SG. 1. Hallek M, Bergsagel PL, Anderson KC (1998). Multiple myeloma: increasing evidence for a multistep transformation process. Blood 91: 3–21. 2. Hussein MA, Juturi JV, Lieberman I (2002). Multiple myeloma: present and future. Curr Opin Oncol 14: 31–35. 2. Hussein MA, Juturi JV, Lieberman I (2002). Multiple myeloma: present and future. Curr Opin Oncol 14: 31–35. 1. Hallek M, Bergsagel PL, Anderson KC (1998). Multiple myeloma: increasing evidence for a multistep transformation process. Blood 91: 3–21. Supporting Information In D, kinetics of MMP-9 and -2 activities in supernatants from healthy donors (HD); zymographic analyses, upper panels; densitometry, lower panels. (TIF) g q Specialized functions of MM cells and microenvironment could be assessed in supernatants from MM explants cultured in the RCCSTM Bioreactor. b2 microglobulin, VEGF and Ang-2, which are released by MM cells, positively and significantly correlated with each other, as already demonstrated in MM sera [45]. Moreover, all factors were reduced upon culture with Bortezomib, concomitantly with PC death. Members of the MMPs family are also expressed in MM microenvironment and are implicated in tumor growth and dissemination, angiogenesis and development of osteolytic lesions. In particular, MMP-9 and possibly MMP-2 are synthesized by MM cells, which also contribute to the activation of the latent forms [27,46]. Accordingly, MMP-9 and -2 were expressed and increased over-time in supernatants; notably, the active forms were detectable during culture, indicating the existence of efficient cell-to-cell interactions. MMP-9 and -2 activities were also found in supernatants from HD, as reported in individuals with hip fractures [47] where they are implicated in fracture healing; however, their levels did not increase over time, neither the active forms could be distinguished (Fig. S3 D). Variations in MMP levels in response to Bortezomib treatment overall mirrored that of MM cells; however, MMPs appeared only slightly affected, supporting the notion that their production and activities result from additional cellular components inside MM microenvironment, including EC, BMSC, and osteoclasts [27,34,35]. Finally, determination of b2 microglobulin levels in patients’sera and in supernatants from Bioreactor disclose an overall concordance in the response to Bortezomib treatment; these data, albeit preliminary given the limited number of patients analyzed and the heterogeneity of Bortezomib-based therapies applied, suggest the possibility to exploit the model on a larger series to predict sensitivity to drugs in individual patients. TM Figure S4 Isolated MM cells from Patient 4 are sensitive to Bortezomib in vitro. A: MM cells isolated from a skull lesion were treated for 24 hrs with Bortezomib (50 nM). 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https://openalex.org/W4309692566	https://www.researchsquare.com/article/rs-2243522/latest.pdf	English	null	Identification of Potential Biomarkers for Colorectal Cancer Using Bioinformatics Analysis	Research Square (Research Square)	2,022	cc-by	4,766	Identification of Potential Biomarkers for Colorectal Cancer Using Bioinformatics Analysis Jiangxi University of Traditional Chinese Medicine Affiliated Hospital mingjun xie Identification of Potential Biomarkers for Colorectal Cancer Using Bioinformatics Analysis an luo Jiangxi University of Traditional Chinese Medicine https://orcid.org/0000-0002-4202-7098 henglang xu Jiangxi University of Traditional Chinese Medicine Affiliated Hospital mingjun xie Jiangxi University of Traditional Chinese Medicine Affiliated Hospital ke hu Jiangxi University of Traditional Chinese Medicine Affiliated Hospital yiping jiang Jiangxi University of Traditional Chinese Medicine Affiliated Hospital jia hu (  hujia2@jxutcm.edu.cn ) https://orcid.org/0000-0003-2387-1557 Research Keywords: colorectal cancer, bioinformatics analysis, differentially expressed genes, hub genes, survival analysis Posted Date: November 22nd, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2243522/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Identification of Potential Biomarkers for Colorectal Cancer Using Bioinformatics Analysis an luo Jiangxi University of Traditional Chinese Medicine https://orcid.org/0000-0002-4202-7098 henglang xu Jiangxi University of Traditional Chinese Medicine Affiliated Hospital mingjun xie Jiangxi University of Traditional Chinese Medicine Affiliated Hospital ke hu Jiangxi University of Traditional Chinese Medicine Affiliated Hospital yiping jiang Jiangxi University of Traditional Chinese Medicine Affiliated Hospital jia hu (  hujia2@jxutcm.edu.cn ) https://orcid.org/0000-0003-2387-1557 Research Keywords: colorectal cancer, bioinformatics analysis, differentially expressed genes, hub genes, survival analysis Posted Date: November 22nd, 2022 DOI: https://doi.org/10.21203/rs.3.rs-2243522/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Results We obtained 756 DEGS (254 upregulated genes and 502 downregulated genes) from the GSE25070 dataset, and DEGs were mainly enriched in inflammatory response, neutrophil chemotaxis, and cytokine- cytokine receptor. Ten hub genes were identified, including five upregulated genes (VEGFA, IL1B, MMP9, CXCL8, and CCND1) and five downregulated genes (MAPK3, ADH1A, SLC26A3, ADH1C, and UGT1A8). Five upregulated genes were highly expressed in CRC patients, and IL1B and CXCL8 genes were significantly associated with overall survival in colorectal cancer patients, and high expression of IL1B and CXCL8 had a greater survival advantage. Conclusion IL1B and CXCL8 are potential biomarkers for CRC. Methods We first identified differentially expressed genes (DEGs) in 26 colorectal tumor tissue samples and 26 matched non-tumor tissue samples in the GSE25070 dataset. We then performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs using the Database for Annotation Visualization and Integrated Discovery (DAVID). We further constructed protein- protein interaction (PPI) networks of DEGs using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database and screened 10 hub genes using Cytoscape software. GO and KEGG enrichment analysis of hub genes was done by the WEB-based GEne SeT AnaLysis Toolkit (WebGestalt). Finally, we analyzed the expression levels and survival of hub genes using the Gene Expression Profiling Interactive Analysis (GEPIA) database. Background Colorectal cancer (CRC) is the most common malignant tumor of the intestine, and its incidence and mortality rate are at the forefront. Early diagnosis and intervention of CRC is of great significance. however, there is a lack of precise diagnostic biomarkers. We aim to explore potential biomarkers for CRC and provide a new treatment idea for CRC. Research License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Page 1/17 Page 1/17 Page 1/17 Microarray Data We first downloaded the microarray expression data of GSE25070 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE25070) from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo). The GSE25070 dataset is based on the GPL6883 platform (Illumina HumanRef-8 v3.0 expression beadchip), and it includes 26 colorectal tumor tissue samples and 26 matched adjacent non-tumor tissue samples. We then further processed the GSE25070 dataset in Microsoft Excel. Introduction Cancer remains the most threatening disease to human beings in the 21st century. Colorectal cancer is one of the most common malignancies of the digestive system, and the Global Cancer Statistics 2020 report(1) shows that colorectal cancer is at the top of the list in terms of incidence and mortality. Early Page 2/17 colorectal cancer may not have obvious symptoms, and when most patients present with complaints of hematochezia, it usually indicates that the disease has progressed to intermediate to advanced stages and metastases are present(2). Most colorectal cancers evolve from polyps, especially adenomatous polyps(3, 4). Unfortunately, many countries and regions do not offer colonoscopy screening as a routine medical examination, leading to this catastrophic event. Currently, serum carcinoembryonic antigen (CEA) is the most commonly used and important diagnostic marker for colorectal cancer(5, 6), but it lacks specificity and sensitivity. Therefore, there is an urgent need to explore new biomarkers to provide new ideas for the treatment of CRC (7–9). To date, a variety of biomarkers are considered to be associated with survival prognosis in CRC. Zong confirmed that CRC patients with high expression of FOXD1 and Plk2 had a worse prognosis by constructing a Nomogram model(10). It was reported that ATP2A1 is a potential pathogenic factor, and CRC patients with high expression of ATP2A1 have a low survival rate(11). It has also been claimed that CX3CR1 promotes cell proliferation and migration of CRC cells by regulating macrophage polarization(12). However, few studies have explored the interaction of biomarkers in CRC through comprehensive analysis. This study aimed to identify potential biomarkers of CRC by bioinformatics analysis. The dataset GSE25070 was used to screen DEGs between colorectal tumor tissues and matched normal adjacent tissues and to perform GO function and KEGG pathway enrichment analysis on DEGs. The STRING database was then used to construct a PPI network of DEGs, and Cytoscape software was used to identify the hub genes of DEGs. Using the WebGestalt online tool, we perform GO and KEGG pathway enrichment analysis of the hub genes. Finally, we performed expression and survival analysis of hub genes using the GEPIA 2 database. Our study identified two potential biomarkers involved in CRC progression, which could be new therapeutic targets for CRC. Analysis Of Go Functional And Kegg Pathway Enrichment Using the DAVID online database (http://david.ncifcrf.gov/tools.jsp), we analyzed GO and KEGG pathway enrichment analyses for 254 upregulated genes and 504 downregulated genes, respectively. GO analysis included biological processes (BP), cellular components (CC), and molecular functions (MF). False discovery rate (FDR) < 0.05 was statistically significant. Identification of DEGs By analyzing the GEO2R online tool in the GEO database, we obtained 756 DEGs from the GSE25070 dataset, including 254 upregulated genes and 502 downregulated genes (Table 1), and visualized the results of the DEGs as a volcano plot (Fig. 1a). Heatmaps of DEGs was plotted using Heml 1.0.3.7 software (Fig. 1b). Ppi Network Analysis And Identification Of Hub Genes To further explore the interaction relationships between DEGs, we used the STRING database (https://string-db.org/) to construct a protein-protein interaction (PPI) network, and Cytoscape 3.9.0 software was used to visualize the PPI results. Meanwhile, we used the cytoHubba plugin in this software to screen the upregulated and downregulated genes for hub genes separately, and the top five genes in terms of degree value were identified as hub genes. Finally, GO and KEGG enrichment analysis was performed for 10 hub genes using the WebGestalt online tool (http://www.webgestalt.org/), and P < 0.05 was statistically significant. Expression And Survival Analysis Of Hub Genes (Dup: Abstract ?) We used the GEPIA 2 database (http://gepia2.cancer-pku.cn) to compare the expression of 10 hub genes between CRC patients and normal patients. We then further explored survival analysis of hub genes, including overall survival and disease-free survival. We divided patients into high and low expression groups based on median hub gene expression and calculated hazards ratios (HR) and their associated 95% confidence intervals (CI) and log-rank test P values. P < 0.05 was considered to be statistical significance. Degs Analysis To further identify DEGs in 26 colorectal tumor tissue samples and 26 matched adjacent non-tumor tissue samples, we used the GEO2R online tool in the GEO database for analysis. Adjusted p-values < 0.05 and \|logFC\| > 1 were set as screening parameters. DEGs with logFC > 0 were identified as upregulated Page 3/17 Page 3/17 genes and those with logFC < 0 were considered as downregulated genes. Heatmaps of the DEGs were drawn by Heml 1.0.3.7 software. Page 4/17 We used the DAVID database to obtain GO functional and KEGG pathway enrichment analyses of DEGs. The GO analysis revealed that 254 upregulated genes were primarily involved in inflammatory response, extracellular matrix organization, and neutrophil chemotaxis biological processes (Fig. 2a, Table 2), whereas 502 downregulated genes were primarily involved in cadmium ion response, retinol metabolic process, and cellular zinc ion homeostasis (Fig. 2b, Table 2). For cellular fractions, upregulated genes were mainly enriched in the extracellular space, extracellular region, and extracellular matrix (Fig. 2a, Table 2), while downregulated genes were enriched in the extracellular exosome, apical plasma membrane, and extracellular space (Fig. 2b, Table 2). In terms of molecular function, the results showed that upregulated genes were mainly enriched in extracellular matrix structural constituent, chemokine activity, and extracellular matrix structural constituent conferring tensile strength (Fig. 2a, Table 2), while the downregulated genes were mainly enriched in structural constituent of muscle, steroid dehydrogenase activity, and retinol dehydrogenase activity (Fig. 2b, Table 2). KEGG pathway enrichment analysis showed that upregulated genes were mainly involved in Rheumatoid arthritis, IL-17 signaling pathway, and Cytokine-cytokine receptor interaction (Fig. 2c, Table 3), while downregulated genes were mainly involved in Metabolic pathways, Drug metabolism - cytochrome P450, and Bile secretion (Fig. 2d, Table 3). Analysis Of Ppi Networks And Hub Genes Using the STRING database, we obtained PPI networks for 254 upregulated genes and 502 downregulated genes. The PPI network for the upregulated gene contains 203 nodes and 1258 edges, and the PPI network for the downregulated gene includes 400 nodes and 1146 edges. We visualized the results using Cytoscape 3.9.0 software (Fig. 3a-b). We also screened 5 hub genes each for upregulated and downregulated genes using the cytoHubba plugin (Fig. 3c-d), including VEGFA, IL1B, MMP9, CXCL8, CCND1, MAPK3, ADH1A, SLC26A3, ADH1C, and UGT1A8. Specific information about the 10 hub genes is shown in Table 4. We further performed GO and KEGG enrichment analysis on 10 hub genes using WebGestalt online tool. GO enrichment analysis showed that the 10 hub genes were mainly enriched in cytokine-mediated signaling pathway, leukocyte migration, myeloid leukocyte migration, and positive regulation of cell migration for BP (Fig. 4a, Table 5); and secretory granule, secretory vesicle, and pseudopodium for CC (Fig. 4b, Table 5); and alcohol dehydrogenase activity, zinc-dependent, alcohol dehydrogenase (NAD) activity, retinol dehydrogenase activity, and molecular function regulator for MF (Fig. 4c, Table 5). KEGG pathway analysis revealed 10 hub genes mainly involved in bladder cancer, AGE-RAGE signaling pathway in diabetic complications, IL-17 signaling pathway, and human cytomegalovirus infection (Fig. 4d, Table 6). Expression And Survival Analysis Of Hub Genes Page 5/17 We performed an analysis using the GEPIA 2 database to further compare the expression of the 10 hub genes between CRC patients and normal patients. The findings revealed that 5 upregulated genes (VEGFA, IL1B, MMP9, CXCL8, and CCND1) were significantly more expressed in colorectal cancer than in normal controls, while 5 downregulated genes (MAPK3, ADH1A, SLC26A3, ADH1C, and UGT1A8) were the inverse (Fig. 5a). Next we performed survival analysis for the 5 upregulated genes, including overall survival and disease- free survival (Fig. 5b-c). The results showed that IL1B and CXCL8 genes were significantly associated with OS in colorectal cancer patients (p < 0.05), and Patients with high expression of IL1B and CXCL8 had a greater survival advantage (Fig. 5b), so we tentatively speculate that IL1B and CXCL8 are potential biomarkers of CRC. Discussion Colorectal cancer is a problem that deserves global attention. The number of diagnosed cases(13) in the United States exceeded 150,000 in 2022, and the incidence of CRC is increasing in China. The number of CRC deaths worldwide(14) is expected to rise significantly by 2035 if early detection services and specialized care are lacking. Here, we obtained 254 upregulated genes and 502 downregulated genes by analyzing the GSE25070 dataset. And these DEGs were analyzed for GO and KEGG pathway enrichment, which revealed that DEGs were mainly enriched in inflammatory response, neutrophil chemotaxis, and cytokine-cytokine receptor interaction pathway. More critically, we screened for hub genes and obtained IL1B and CXCL8 by further analysis of hub genes, demonstrating that they are closely associated with CRC progression and survival prognosis. IL1B is a member of the inflammatory cytokine interleukins(15), and numerous previous studies have shown that inflammation plays a crucial role in the development of tumors(16–19). IL1B has been reported to be associated with the development and metastasis of a variety of tumors, including non-small cell lung cancer(20), cervical cancer(21), and breast cancer(22). Although high expression of pro-inflammatory cytokines seems to be detrimental to the survival prognosis of CRC patients, studies have claimed that IL1B gene rs1143623 and rs1143634 polymorphisms are associated with reduced risk and better survival in CRC patients(23). CXCL8 (also known as interleukin-8) is a member of the CXC chemokine family(24), and CXC has been identified as a potential therapeutic target for a variety of cancers as a fundamental regulator of leukocyte directed migration(25–27). Numerous studies have demonstrated that CXCL8 is highly expressed in CRC and is closely associated with CRC progression(28– 30). However, does high expression of CXCL8 predict a poor prognosis for CRC patients? This remains a controversial question, and there are studies(31) confirming a greater survival advantage of high CXCL8 expression in CRC patients, which is consistent with our findings. In addition to inflammatory response, our study showed that DEGs are also mainly enriched in neutrophil chemotaxis and cytokine-cytokine receptor interaction pathway. Tumors orchestrate neutrophil recruitment by releasing neutrophil-associated chemokines(32), and CXCL8, a neutrophil-specific Page 6/17 Page 6/17 Page 6/17 chemokine, is elevated in the CRC tumor microenvironment and promotes angiogenesis, invasion, and metastasis(33–35). Numerous studies have reported significant enrichment of the cytokine-cytokine receptor interaction pathway in CRC(36–38). Conclusion In conclusion, based on bioinformatics analysis of DEGs in CRC samples and matched adjacent normal tissue samples, we finally identified 2 hub genes (IL1B and CXCL8). Our study suggests that IL1B and CXCL8 may be potential biomarkers for colorectal cancer. Discussion It has been shown that cytokine-cytokine receptor interaction was significantly and positively correlated with tumor metastatic areas, and the enrichment score of the cytokine-cytokine receptor interaction pathway decreased after treatment with IP6 in an in situ transplantation model of CRC mice, thus interfering with CRC metastasis(39). In summary, our study suggests that both IL1B and CXCL8 are associated with inflammatory response, neutrophil chemotaxis, and cytokine-cytokine receptor interaction pathway. However, our study has some limitations. The sample size of the GSE25070 dataset is not large enough, and we need experiments to further validate our results. Therefore, our findings should be reserved for further confirmation in future studies. Abbreviations CRC colorectal cancer DEGs differentially expressed genes GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes DAVID Database for Annotation Visualization and Integrated Discovery PPI protein-protein interaction STRING Search Tool for the Retrieval of Interacting Genes/Proteins WebGestalt WEB-based GEne SeT AnaLysis Toolkit GEPIA Gene Expression Profiling Interactive Analysis CEA serum carcinoembryonic antigen GEO Page 7/17 Gene Expression Omnibus BP biological processes CC cellular components MF molecular functions. Gene Expression Omnibus BP biological processes CC cellular components MF molecular functions. Authors’ Contributions AL and JH participated in the design of this project. AL and JH analyzed the experimental data. AL drafted the manuscript. KH, HLX, MJX, YPJ, and JH contributed to the revision of the manuscript. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Acknowledgments Not applicable. Funding This study was supported by the Jiangxi Province Traditional Chinese Medicine Young and Middle-aged Backbone Talents (Third Batch) Training Program (Ganzhong Medicine Kejiaozi [2021] No. 2). Consent for publication Not applicable. Ethics approval and consent to participate This study does not contain any studies with human participants or animals. Availability of data and materials GSE25070 dataset (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE25070). Jia Hu https://orcid.org/0000-0003-2387-1557 Jia Hu https://orcid.org/0000-0003-2387-1557 References 1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: A Cancer Journal for Clinicians. 2021;71(3):209 – 49. 2. Bendardaf R, El-Serafi A, Syrjänen K, Collan Y, Pyrhönen S. 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Gene expression and methylation profiles identified CXCL3 and CXCL8 as key genes for diagnosis and prognosis of colon adenocarcinoma. J Cell Physiol. 2020;235(5):4902–12. 32. Bonavita O, Massara M, Bonecchi R. Chemokine regulation of neutrophil function in tumors. Cytokine Growth Factor Rev. 2016;30:81–6. 32. Bonavita O, Massara M, Bonecchi R. Chemokine regulation of neutrophil function in tumors. Cytokine Growth Factor Rev. 2016;30:81–6. 33. Schimek V, Strasser K, Beer A, Göber S, Walterskirchen N, Brostjan C, et al. Tumour cell apoptosis modulates the colorectal cancer immune microenvironment via interleukin-8-dependent neutrophil recruitment. Cell Death Dis. 2022;13(2):113. 33. Tables Table 1 to 6 are available in the Supplementary Files section. Page 11/17 Figures Figure 1 Volcano plots and heatmaps of DEGs expression in the GSE25070 dataset. a Volcano plots, red dots represent upregulated genes, green dots represent downregulated genes, and black dots represent no differentially expressed genes. b Heatmaps of DEGs Figure 1 Figure 1 Volcano plots and heatmaps of DEGs expression in the GSE25070 dataset. a Volcano plots, red dots represent upregulated genes, green dots represent downregulated genes, and black dots represent no differentially expressed genes. b Heatmaps of DEGs Page 12/17 Page 12/17 Page 12/17 Figure 2 Functional enrichment analysis of DEGs. a GO functional analysis of upregulated genes. b GO functiona analysis of downregulated genes. c KEGG pathway analysis of upregulated genes. d KEGG pathway analysis of downregulated genes Figure 2 Figure 5 Expression and Survival Analysis of Hub Genes. a The expression of 10 hub genes between CRC patients and normal patients. b Overall survival analysis of 5 upregulated genes. c Disease-free survival analysis of 5 upregulated genes Figure 2 Functional enrichment analysis of DEGs. a GO functional analysis of upregulated genes. b GO functional analysis of downregulated genes. c KEGG pathway analysis of upregulated genes. d KEGG pathway analysis of downregulated genes Functional enrichment analysis of DEGs. a GO functional analysis of upregulated genes. b GO functional analysis of downregulated genes. c KEGG pathway analysis of upregulated genes. d KEGG pathway analysis of downregulated genes Page 13/17 Page 13/17 Figure 3 Analysis of PPI Networks and Hub Genes. a PPI network for upregulated genes. b PPI network for downregulated genes. c 5 hub genes for upregulated genes. d 5 hub genes for downregulated genes Figure 3 Analysis of PPI Networks and Hub Genes. a PPI network for upregulated genes. b PPI network for downregulated genes. c 5 hub genes for upregulated genes. d 5 hub genes for downregulated genes Figure 3 Analysis of PPI Networks and Hub Genes. a PPI netw Figure 3 Analysis of PPI Networks and Hub Genes. a PPI network for upregulated genes. b PPI network for downregulated genes. c 5 hub genes for upregulated genes. d 5 hub genes for downregulated genes Analysis of PPI Networks and Hub Genes. a PPI network for upregulated genes. b PPI network for downregulated genes. c 5 hub genes for upregulated genes. d 5 hub genes for downregulated genes Page 14/17 Figure 4 GO and KEGG enrichment analysis of 10 hub genes. a Biological processes. bCellular components. c Molecular function. d KEGG pathways Figure 4 Figure 4 Figure 4 GO and KEGG enrichment analysis of 10 hub genes. a Biological processes. bCellular components. c Molecular function. d KEGG pathways GO and KEGG enrichment analysis of 10 hub genes. a Biological processes. bCellular components. c Molecular function. d KEGG pathways Page 15/17 Page 15/17 Figure 5 Expression and Survival Analysis of Hub Genes. a The expression of 10 hub genes between CRC patients and normal patients. b Overall survival analysis of 5 upregulated genes. c Disease-free survival analysis of 5 upregulated genes Figure 5 Table3.xls Table4.xls Table5.xls Table6.xls Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Table1.xls Table2.xls Page 16/17 Table3.xls Table4.xls Table5.xls Table6.xls Page 17/17
W1913043303.txt	http://www.scielo.br/pdf/coluna/v8n4/v8n4a07.pdf	en		Intrathecal morphine in lumbar spine fusion	null	2,009	cc-by	2,729	ARTIGO ORIGINAL / ORIGINAL ARTICLE Uso de morfina intratecal en artrodesis lumbar Uso da morfina intratecal na artrodese lombar Intrathecal morphine in lumbar spine fusion Ronald Schulz Ibaceta1 Nicolás Macchiavello Cornejo1 Sergio Ramirez2 Jaime Escobar3 Marcos Ganga Villagrán1 Hermann Schulz4 RESUMEN Objetivo: determinar la eficacia y la seguridad del uso de morfina intratecal, en bajas dosis, en pacientes sometidos a cirugía de instrumentación y artrodesis lumbar. Métodos: estudio prospectivo, randomizado, ciego y controlado. Fueron utilizados dos grupos de pacientes: Grupo Estudio, que recibió morfina intratecal al final de su cirugía, y Grupo Control que sólo recibió el protocolo de analgesia estándar. Resultados: se encontraron diferencias significativas en la escala visual análoga (EVA) entre los dos grupos a las 12 horas postoperatorias. La EVA en reposo promedio del Grupo Estudio fue de 2,15 cm y el del Grupo Control, 5 cm (p=0,013). En actividad, el Grupo Estudio presentó una EVA promedio de 4,36 cm, y el Grupo Control 6,9 cm (p=0,029). No se encontraron diferencias en relación a las complicaciones entre los dos grupos. Conclusión: el uso de morfina intratecal, en bajas dosis, es seguro y efectivo en el control del dolor en las primeras 12 horas postoperatorias en cirugía de artrodesis lumbar. RESUMO Objetivo: determinar a eficácia e a seguridade do uso da morfina intratecal, em baixas doses, em pacientes submetidos à cirurgia de instrumentação e artrodese lombar. Métodos: estudo prospectivo, randomizado, cego e controlado. Foram utilizados dois grupos de pacientes: Grupo Estudo, que recebeu morfina intratecal no final da cirurgia e o Grupo Controle, que recebeu somente o protocolo de analgesia padrão. Resultados: foram encontradas diferenças significativas na escala visual analógica (EVA) entre os dois grupos às 12 horas pós-operatórias. A EVA em repouso, em média, do Grupo Estudo foi de 2,15 cm e do Grupo Controle, 5 cm (p=0,013). Durante atividade, o Grupo Estudo apresentou uma EVA de aproximadamente 4,36 cm e no Grupo Controle, 6,9 cm (p=0,029). Não foram encontradas diferenças com relação às complicações entre os dois grupos. Conclusão: o uso de morfina intratecal, em baixas doses, foi seguro e efetivo no controle da dor nas primeiras 12 horas pós-operatórias na cirurgia de artrodese lombar. Study carried out at Medical School, Universidad de Chile, Chile. Spine Unit. Orthopaedics Department. Hospital Clínico Universidad de Chile, Chile. Intern. Medical School, Universidad de Chile, Chile. Anesthesiologist. Anethesiology Department. Hospital Clínico Universidad de Chile, Chile. 4 Medical Student. Medical School, Universidad de los Andes, Chile. 1 2 3 Recibido: 21/7/2009 Acepto: 30/10/2009 COLUNA/COLUMNA. 2009;8(4):386-389 ABSTRACT Objective: to determine the efficacy and safety of low-dose intrathecal morphine use in lumbar instrumented arthrodesis. Methods: prospective, randomized, blind and controlled study, comparing two groups of patients, with and without the administration of intrathecal morphine at the end of surgery. Results: statistically significant differences were found in the visual analogue score (VAS) between the two groups, 12 hours after surgery. Average VAS (at rest) in the Study Group was 2.15 cm versus 5 cm in the Control Group (p=0.013). In activity, average, VAS in the Study Group was 4.36 cm and in the Control Group, 6.9 cm (p=0.029). No differences were found when comparing complication rates. Conclusion: the use of low-dose intrathecal morphine in instrumented lumbar arthrodesis, for postoperative pain management, is safe and effective. Uso de morfina intratecal en artrodesis lumbar DESCRIPTORES: Fusión vertebral/instrumentación; Morfina/administración & dosificación; Morfina/ efectos adversos; Dolor postoperatorio/etiología; Dolor postoperatorio/ prevención & control 387 DESCRITORES: Fusão vertebral/ instrumentação; Morfina/ administração & dosagem; Morfina/efeitos adversos; Dor pós-operatória/etiologia; Dor pós-operatória/prevenção & controle INTRODUCCIÓN El manejo del dolor postoperatorio es tal vez uno de los desafíos más importantes a los que se ve enfrentando el equipo médico de los pacientes que han sido sometidos a una cirugía. Este no sólo repercute sobre la sensación de bienestar del paciente, si no que también se asocia a una mayor morbi-mortalidad1, puede alterar la respuesta inmune2 y, en pacientes senescentes, aumenta el riesgo de delirio3. Existen distintas alternativas para el manejo del dolor postoperatorio; entre ellas hay el uso de antiinflamatorios no esteroidales (AINES) y opioides sistémicos, anestésicos y opioides via epidural o intratecal, así como bloqueos regionales. Cada una de éstas alternativas presenta las ventajas y desventajas propias del medicamento y la vía por la que se utiliza. El uso de opioides por vía sistémica puede provocar sedación, náuseas, vómitos, depresión respiratoria, reducción de la motilidad intestinal, adicción, retención urinaria, entre otras4. El uso de anestésicos regionales conlleva el riesgo de una alteración neurológica que puede interferir con el diagnóstico precoz y el manejo de complicaciones perioperatorias. En el caso de la morfina, diferentes estudios han demostrado que sus receptores se encuentran localizados en el tejido cerebral y en la médula espinal5-7 y que su efecto analgésico estaría mediado por receptores pre- y post-sinápticos en las células de la asta dorsal y a niveles supraespinales. Específicamente en la cirugía de artrodesis lumbar, la exposición de la columna por abordaje posterior facilita la colocación de morfina intratecal, la que podría actuar directamente sobre las astas dorsales en la zona operatoria. La vía intratecal parecería ser superior a la peridural, pero sus complicaciones aumentan en la medida que aumenta la dosis utilizada. Es por esto que nos planteamos las siguientes interrogantes: ¿es la morfina intratecal eficaz, a bajas dosis, en el control del dolor postoperatorio?; ¿es segura?. Mediante el presente estudio, se buscó determinar la eficacia y la seguridad del uso intratecal de morfina en pacientes sometidos a cirugía de columna. MÉTODOS Estudio prospectivo, randomizado, ciego y controlado. Veinte nueve pacientes sometidos a cirugía de instrumentación y artrodesis lumbar fueron divididos en dos grupos: el primer grupo, denominado Grupo Estudio, KEYWORDS: Spinal fusion/ instrumentation; Morphine/ administration & dosage; Morphine/adverse effects; Pain, postoperative/etiology; Pain, postoperative / prevention & control recibió morfina intratecal al finalizar su cirugía, antes del cierre de la herida; y el segundo, denominado Control, no. La selección de los pacientes asignados a cada grupo fue mediante randomización al azar. Los pacientes no tuvieron acceso a información que les permitiera saber a qué grupo correspondían. Los citerios de exclusión fueron: embarazo, lactancia, infección, paresia de extremidades inferiores, antecedentes de alergia y durotomía intraoperatoria. Ambos grupos recibieron la misma analgesia postoperatoria estándar (ketoprofeno 300 mgs + tramadol 200 mgs + droperidol 2,5 mgs en infusión continua endovenosa) y de rescate (PCA de morfina en dosis de 1 mgs con lockout de ocho a diez minutos). El Grupo Estudio recibió, además, un bolo de entre 80 y 150 gamas de morfina intratecal, diluídos en solución fisiológica (volumen total 3 mL) en el intraoperatorio, entre L3 y L5, bajo visión directa, utilizando un trócar punta de lápiz de 27 G. Un evaluador independiente, ciego, controló el dolor en actividad y en reposo mediante la escala visual análoga (EVA) (0 a 10 cm) a las 12, 24 y 48 horas postoperatorias. Además, se registraron las complicaciones asociadas a la morfina y a la punción lumbar. Los resultados fueron analizados utilizando el software SPSS 11.0.4, versión para Macintosh. Se calcularon promedios, desviación estándar, y se utilizaron los testes de Wilcoxon y Mann Whitney, con un nivel de significancia igual o menor a 0,05. RESULTADOS Trece pacientes (ocho hombres y cinco mujeres) fueron incluidos en el Grupo Estudio y 16 (diez hombres y seis mujeres) en el Grupo Control. Las edades fluctuaron entre los 13 y los 71 años, siendo el promedio 49 en el Grupo Estudio y 44 en el Control. Las patologías fueron: espondilolistesis (24%), hernias del núcleo pulposo (21%), fracturas (14%), osteocondrosis (14%), estenosis (10%), síndrome post-nucleotomia (10%) y escoliosis (7%). La distribución según tipo de cirugía se resume en la Tabla 1. En relación al dolor postoperatorio, los resultados mostraron que en reposo a las 12 horas, la EVA promedio del Grupo Estudio fue de 2,15 cm, y del Grupo Control fue 5 cm (p=0,013). En actividad, el Grupo Estudio presentó una EVA promedio de 4,36 cm y el Grupo Control, 6,9 cm (p=0,029). COLUNA/COLUMNA. 2009;8(4):386-389 Schulz IR, Macchiavello CN, Ramirez S, Escobar J, Ganga VM 388 TABLA 1 – Distribución por tipo de cirugía Origen Control Caso Total Cirugía Nucleotomía 1 1 2 Espondilodesis posterior 3 2 5 Total PLIF 1 1 2 Reemplazo vertebral 3 1 4 TLIF 8 8 16 16 13 29 TABLA 2 – Complicaciones postoperatorias Origen Náuseas Control Caso 3 3 6 Total Complicaciones Prurito Retención urinaria 1 1 1 1 EVA 6 5 4 3 2 1 0 Series 2 5 4,06 12 2,88 2,46 2,62 2,15 24 Horas 48 Figura 1 Curva de dolor en reposo desde las 12 a las 48 horas de operado Dolor en actividad EVA Morfina 8 7 6 5 4 3 2 1 0 Control 1 2 1 12 2 24 Horas 3 3 48 Figura 2 Curva de dolor en actividad desde las 12 a las 48 hrs de operado En los siguientes controles, las medias de dolor fueron siempre menores en el grupo tratado con morfina, pero no alcanzaron la significancia estadística (Figuras 1 y 2). Las complicaciones relacionadas con el uso de la morfina fueron similares en ambos grupos e incluyeron: náuseas, retención urinaria, hipotensión, prurito, cefalea y delirio (Tabla 2). No se registraron complicaciones relacionadas con la punción. COLUNA/COLUMNA. 2009;8(4):386-389 Delirio 1 1 1 1 Cefalea 1 DISCUSIÓN Dolor de reposo Series 1 Total Hipotensión 1 5 6 11 En los años 1970 se encontraron receptores de morfina en el tejido cerebral y de la médula espinal5,6. Estudios en animales demostraron las propiedades analgésicas de la morfina usada vía intratecal8,9. Wang y, posteriormente, otros autores demostraron que esta vía de administración en humanos es exitosa4,10. Se piensa que el efecto analgésico es mediado por receptores pre- y post-sinápticos en la sustancia gelatinosa de las células de la asta dorsal espinal, y a niveles supraespinales7,8,11. La vía intratecal para la administración de morfina local en cirugía lumbar es aparentemente superior a la peridural, ya que tendría menor riesgo de infección, no existe dilución del medicamento en el hematoma y no existe pérdida posterior por los drenajes. Asimismo, la distribución del fármaco en el espacio epidural es variable e impredecible, como resultado de la dilución de éste en el hematoma operatorio. En la literatura se describe el uso de dosis de morfina intratecal que varía entre los 250 a 2.500 gamas, siendo la dosis habitual entre 250 y 500 gamas. Ziegeler et al.4 demostraron que dosis de 400 gamas tienen suficiente efecto analgésico, con una baja tasa de complicaciones, ninguna de ellas severa. Debido a que la morfina es un opioide hidrofílico con una alta afinidad por sus receptores, su uso por vía intratecal tiene una latencia clínica de 45 a 75 minutos, y una larga duración (hasta 24 horas)12. El ajustar la dosis según el peso del paciente es controversial. Se ha demostrado que el volumen del líquido cefalorraquídeo es inversamente proporcional al índice de masa corporal, sin embargo existen diversas patologías que pueden modificar este volumen13. Por otra parte, Eisenach et al. pudo demostrar que la distribución de la morfina intratecal no se correlaciona con el volumen del líquido cefalorraquídeo, peso o estatura del paciente14. De este modo, el ajuste de la dosis según peso del paciente no permitiría asegurar un efecto clínico predecible. La relación entre la dosis y el efecto parece tener su techo en las 500 gamas; sobre esta dosis se obtiene el mismo Uso de morfina intratecal en artrodesis lumbar 389 efecto analgésico pero aumentan las complicaciones10,12. Dosis superiores a 1.000 gamas se asocian a complicaciones severas. A la luz de los resultados expuestos, fue demostrado que el uso de dosis bajas de morfina intratecal (80 a 150 gamas), en cirugía de artrodesis lumbar, permite obtener excelentes resultados en el manejo del dolor postoperatorio, especialmente dentro de las primeras 12 horas. En este rango de dosis, las complicaciones asociadas al uso de la morfina no aumentan en forma significativa, en relación a las mismas en pacientes que no la recibieron, lo que da seguridad para la utilización de ésta. Ross et al.15, en un estudio prospectivo, controlado, doble ciego, compararon dosis de morfina intratecal de 125, 250 y 500 gamas, con un placebo. Él publica que dosis de 125 gamas no tienen efecto superior al del placebo. Concluye que en pacientes jóvenes y sanos debieran usarse dosis de 250 a 500 gamas, mientras que en pacientes ancianos o con comorbilidad debiera usarse la dosis de 250 gamas. Sin embargo, el estudio de Ross incluye una serie de diversas técnicas quirúrgicas, lo que en cierta medida resta validez a sus conclusiones. En contraposición a los resultados del presente estudio, Boezaart et al.16, en un estudio en el que compara dosis de morfina de 200, 300 y 400 gamas, observaron que 200 gamas serían insuficientes en el control del dolor. Sin embargo, el estudio de Boezaart et al. carecían de un Grupo Control. En nuestro conocimiento, este trabajo es el primero en demostrar que dosis tan bajas de morfina bastan para controlar el dolor postoperatorio en los pacientes sometidos a artrodesis de la columna lumbar. Futuros estudios debieran investigar si el efecto del fentanyl intratecal es equivalente o superior al de la morfina. Hipotéticamente, por su mayor lipofilicidad, debiera de asociarse a un menor riesgo de depresión respiratoria. Chan et al. reportan que dosis de 15 gamas tienen efecto superior al de la morfina, ambos usados por vía intratecal17. CONCLUSIÓN El uso de morfina intratecal en bajas dosis (80 a 150 gamas), en cirugía de artrodesis lumbar, es una herramienta segura y efectiva en el control del dolor en las primeras 12 horas postoperatorias. La duración de su efecto permite reducir el dolor desde el postoperatorio inmediato y hasta las primeras 12 a 24 horas, reduciendo así la necesidad de analgesia adicional durante este período, que usualmente es el de mayor dolor. No obstante, debido al riesgo de extensión rostral de la morfina intratecal, los pacientes debieran ser controlados en una Unidad de Cuidados Intensivos, al menos por 12 horas, por el riesgo de depresión respiratoria. REFERENCIAS 1. Tsui SL, Law S, Fok M, Lo JR, Ho E, Yang J, Wong J. Postoperative analgesia reduces mortality and morbidity after esophagectomy. Am J Surg. 1997;173(6):472-8. 2. Kremer MJ. Surgery, pain, and immune function. CRNA. 1999;10(3):94-100. 3. Fong HK, Sands LP, Leung JM. The role of postoperative analgesia in delirium and cognitive decline in elderly patients: a systematic review. Anesth Analg. 2006;102(4):1255-66. 4. Ziegeler S, Fritsch E, Bauer C, Mencke T, Müller BI, Soltesz S, Silomon M. Therapeutic effect of intrathecal morphine after posterior lumbar interbody fusion surgery: a prospective, double-blind, randomized study. Spine (Phila Pa 1976). 2008;33(22):2379-86. 5. Lamotte C, Pert CB, Snyder SH. Opiate receptor binding in primate spinal cord: distribution and changes after dorsal root section. Brain Res. 1976;112(2):407-12. 6. Pert CB, Snyder SH. Opiate receptor: demonstration in nervous tissue. Science. 1973;179(77):1011-4. 7. Yaksh TL. Opioid receptor systems and the endorphins: a review of their spinal organization. J Neurosurg. 1987;67(2):157-76. 8. Yaksh TL, Rudy TA. Analgesia mediated by a direct spinal action of narcotics. Science. 1976;192(4246):1357-8. 9. Wang JK. Analgesic effect of intrathecally administered morphine. Reg Anaesth Pain Med. 1977;2(3):3-8. 10.Wang JK, Nauss LA, Thomas JE. Pain relief by intrathecally applied morphine in man. Anesthesiology. 1979;50(2):149-51. 11. Besson JM, Chaouch A. Peripheral and spinal mechanisms of nociception. Physiol Rev. 1987;67(1):67-186. Review. 12.Rathmell JP, Lair TR, Nauman B. The role of intrathecal drugs in the treatment of acute pain. Anesth Analg. 2005;101 (5 Suppl):S30-43. 13.Sullivan JT, Grouper S, Walker MT, Parrish TB, McCarthy RJ, Wong CA. Lumbosacral cerebrospinal fluid volume in humans using three-dimensional magnetic resonance imaging. Anesth Analg. 2006;103(5):1306-10. 14.Eisenach JC, Hood DD, Curry R, Shafer SL. Cephalad movement of morphine and fentanyl in humans after intrathecal injection. Anesthesiology. 2003;99(1):166-73. 15.Ross DA, Drasner K, Weinstein PR, Flaherty JF, Barbaro NM. Use of intrathecally administered morphine in the treatment of postoperative pain after lumbar spinal surgery: a prospective, double-blind, placebocontrolled study. Neurosurgery. 1991;28(5):700-4. 16.Boezaart AP, Eksteen JA, Spuy GV, Rossouw P, Knipe M. Intrathecal morphine. Double-blind evaluation of optimal dosage for analgesia after major lumbar spinal surgery. Spine (Phila Pa 1976). 1999;24(11):1131-7. 17.Chan JH, Heilpern GN, Packham I, Trehan RK, Marsh GD, Knibb AA. A prospective randomized doubleblind trial of the use of intrathecal fentanyl in patients undergoing lumbar spinal surgery. Spine (Phila Pa 1976). 2006;31(22):2529-33. Correspondencia Ronald Schulz Servicio de Traumatología Avenida Santos Dumont, 999 – Independencia, Chile Tel.: 56-9-9228-0824 Fax: 56-2-978-8941 E-mail: schulzibaceta@gmail.com COLUNA/COLUMNA. 2009;8(4):386-389
https://openalex.org/W2990088641	https://myukk.org/SM2017/sm_pdf/SM2042.pdf	English	null	High-performance Energy Storage System Using Low-cost Sensors for a Photovoltaic and Battery Hybrid System	Sensors and materials	2,019	cc-by	5,063	Corresponding author: e-mail: chaokh@ncut.edu.tw https://doi.org/10.18494/SAM.2019.2200 High-performance Energy Storage System Using Low-cost Sensors for a Photovoltaic and Battery Hybrid System High-performance Energy Storage System Using Low-cost Sensors for a Photovoltaic and Battery Hybrid System Pi-Yun Chen, Kuei-Hsiang Chao, and Yu-Sheng Tsai Department of Electrical Engineering, National Chin-Yi University of Technology, No. 57, Sec. 2, Zhongshan Rd. Taiping Dist. Taichung 41170, Taiwan (ROC) (Received Novembe 19, 2018; accepted May 7, 2019) Keywords: battery charger, photovoltaic module array, buck converter, maximum power point tracker, two-stage charging strategy Keywords: battery charger, photovoltaic module array, buck converter, maximum power point tracker, two-stage charging strategy In this paper, we present a high-speed battery charger, powered by a photovoltaic (PV) module array, for a LiFePO4 battery as a solar energy storage device. With a battery charging strategy, the presented battery charger involves a buck converter as the core equipped with a simple maximum-power-point (MPP) tracker. Considering complexity reduction and easy hardware implementation, a constant-voltage MPP tracking approach is adopted such that the maximum amount of output power can be delivered to the load in response to an arbitrary change in the level of solar radiation. A two-stage charging strategy, with temperature monitoring in a constant current mode followed by a constant voltage mode, is employed in such a way that the battery charge process can be markedly accelerated, while the damage caused by overcharging can be prevented. Finally, the performance of the proposed energy storage system using low-cost voltage, current, and temperature sensors for a PV and battery hybrid system is validated experimentally. 3691 3691 Sensors and Materials, Vol. 31, No. 11 (2019) 3691–3703 MYU T k Sensors and Materials, Vol. 31, No. 11 (2019) 3691–3703 MYU Tokyo S & M 2042 1. Introduction There is a rapid rise in electricity price as a consequence of the upcoming global oil crisis, and the search for and the development of alterative energy sources, particularly green energy sources, become a critical issue. Solar energy has long been seen as a promising candidate energy source to resolve the threat of oil crisis, but a disadvantage is that the generated solar power cannot be well regulated owing to a number of factors, e.g., time-varying solar radiation, climate uncertainty, and so forth. Hence, it is a significant and urgent issue to improve the performance of solar power generators in practical uses.(1) As a reusable electric device, secondary batteries continue to gain worldwide attention owing to global warming and ecofriendly concerns. In an attempt to reduce the amount of CO2 emission, a great number of research institutes and business entities have made continuous effort to search for alternative energy sources, such as solar power, wind power, and fuel cell, or to improve the conversion of secondary batteries. There is no doubt that Li-ion batteries will ISSN 0914-4935 © MYU K.K. https://myukk.org/ ISSN 0914-4935 © MYU K.K. https://myukk.org/ 3692 Sensors and Materials, Vol. 31, No. 11 (2019) gradually take the place of lead-acid and Ni-hydrogen batteries in the long term; particularly, LiFePO4 batteries rank first among a wide variety of Li-ion batteries owing to their high capacity and long service time.(2) In addition, a LiFePO4 battery does not require a high current charger, indicating that it can be charged by a solar-powered battery charger even at low levels of solar radiation. The performance is validated experimentally only using low-cost voltage, current, and temperature sensors. The output power of a photovoltaic (PV) module is a function of solar radiation, module temperature, and so forth. Owing to efficiency concerns, the maximum power point (MPP) is tracked by a constant-voltage MPP tracking approach herein so as to transfer the maximum amount of power at all events to a LiFePO4 battery through the presented battery charger. A two-stage charging strategy is employed in such a way that the charger works in the constant current mode to speed up the charging of a high-capacity LiFePO4 battery, and then operates in the constant voltage mode so as to keep the battery voltage below the evaporation voltage for safety concern. In this manner, efficiency improvement, charge process acceleration, and an extended battery life are hence achieved. 1. Introduction For the performance testing of this presented two-stage battery charging strategy, high current charge/discharge characteristic curves of a LiFePO4 battery, i.e., plots of the battery voltage, current, and state of charge (SOC) against time, are obtained for the behavioral analysis of batteries. where Iph represents the short-circuit current at a given level of solar radiation, where Iph represents the short-circuit current at a given level of solar radiation, where Iph represents the short-circuit current at a given level of solar radiation, Isat = reverse saturation current of the diode D1, 1 q = charge carried by a single electron (1.6 × 10−19C), Vo = output voltage of the solar cell, A = dielectric constant (1–2), K = Boltzmann’s constant (1.3806505×10−23 JK−1), Rsh = equivalent shunt resistance, Rs = equivalent series resistance, and T = surface temperature of the solar cell [25 °C (298 K)]. 2.1 Solar cell basics A solar cell is a semiconductor device. Over the electrically neutral PN region, electron–hole pairs are generated as a consequence of incident solar energy. Owing to the effect of the electric field across the depletion region, electrons and holes drift across the P–N junction toward the N and P regions, respectively. Illustrated in Fig. 1 is a mathematic model of a solar cell, where Iph represents the short-circuit current at a given level of solar radiation and Rs the contact resistance between the metal electrodes and the semiconductor material. Fig. 1. Circuit model of a PV cell. Fig. 1. Circuit model of a PV cell. Sensors and Materials, Vol. 31, No. 11 (2019) 3693 The output current Io, provided by a solar cell, is given as ( ) exp 1 ph sat q V I R V I R o o s o o s I I I o AKT Rsh + + = − − −               , (1) (1) 2.2 MPP tracking strategies for a PV module array The performance of a PV module array and MPPs demonstrate a strong dependence on exterior factors, e.g., solar radiation, temperature, and so forth. In consideration of efficiency optimization, an MPP tracker is employed to deliver the maximum amount of solar power to the load. Up to now, there have been a great number of published studies on this issue,(3,4) among which the constant voltage, power feedback, perturb and observe, incremental conductance methods, and so on are the most commonly seen approaches, and the constant voltage method is demonstrated as the easiest and simplest way to perform a tracking task. The idea is to make a PV module array work at a specific output voltage. Illustrated in Fig. 2 is a one- parameter family of I–V characteristic curves. As revealed, the MPP voltage demonstrates little dependence on the level of solar radiation. In simple terms, the design complexity of an MPP tracker can be reduced considerably, provided that the tracker is operated at the maximum power voltage specified in the datasheet thereof. On account of the high reliability, high Fig. 2. Family of I–V characteristic curves of a PV module array with the solar radiation level as a parameter. Fig. 2. Family of I–V characteristic curves of a PV module array with the solar radiation level as a parameter. 3694 Sensors and Materials, Vol. 31, No. 11 (2019) stability, and low cost of the constant-voltage MPP tracking approach, this approach is employed herein. 3. Programmable Interface Controller (PIC) Microcontroller PIC series microcontrollers, including 8-, 16-, and 32-bit microcontrollers, are provided by Microchip Technology Inc.(5) As the controller core, a PIC18F8720 microcontroller(6) is employed with the following underlying key features: (1) a high-performance reduced instruction set computing (RISC) CPU, (1) a high-performance reduced instruction set computing (RISC) CPU, (2) linear program memory addressing to 128 kB, linear data memory addressing to 3840 bytes, 1 kB of data EEPROM, up to 10 MIP operation, DC – 40 MHz osc./clock input with PLL active loop filter, (2) linear program memory addressing to 128 kB, linear data memory addressing to 3840 bytes, 1 kB of data EEPROM, up to 10 MIP operation, DC – 40 MHz osc./clock input with PLL active loop filter, (3) a 16-bit external memory interface, address capability of up to 2 MB, (4) a high-current sink/source (25 mA/25 mA), (5) four external interrupt pins, (6) five capture/compare/PWM (CCP) modules, (7) a programmable 16-level low-voltage detection (LVD) module, and (8) a 10-bit, up to 16-channel analog-to-digital converter (A/D). 4.1 Li–iron battery survey Among a wide range of secondary batteries, lithium-ion, particularly Li–iron,(7) batteries gain wide popularity in practical applications owing to their advantages of high energy density, high discharge voltage, low self-discharge rate, no memory effect, and long battery life. LiFePO4 batteries, also known as Li–iron batteries, were proposed in 1997 by Padhi and coworkers(8–10) with LiFePO4 as a positive electrode material. In contrast to conventional LiMn2O4, LiNiO2, and LiCoO2, batteries, a LiFePO4 battery does not contain any precious metals, e.g., Mn, Ni, and Co; hence, it acquires a cost advantage. In addition, it is a high-safety, high-capacity, and ecofriendly energy storage device. As opposed to inherently unsafe LiCoO2 batteries, there exists a strong covalent bond between molecules of positive electrode materials in a LiFePO4 battery. For this reason, the risk of explosion and flame is completely removed during the use of LiFePO4 batteries, a clear advantage over other types of batteries. Moreover, LiFePO4 batteries serve as a high- performance energy storage device, since the requirements of high capacity, zero pollution, low self-discharge rate, long service life (the number of discharge and charge cycles), high coulombic efficiency, light weight, and high safety can be fulfilled. Furthermore, it features high-current charge/discharge. Accordingly, a 48 V 10 Ah EA-480100-C15 LiFePO4 battery is employed as an energy storage device for the presented PV system. The specifications of the Li–iron battery are given in Table 1. 3695 Sensors and Materials, Vol. 31, No. 11 (2019) Table 1 LiFePO4 battery specifications. Name EA-480100-C15 Rated capacity 10 Ah Rated voltage 52.8 V Charging cutoff voltage 58.4 V Discharging cutoff voltage 45.6 V Maximum charge current 10 A Temperature range of charging 0–60 °C Temperature range of discharging −20–70 °C 4.2 PIC microcontroller charging strategies for Li–iron batteries Schematic of a two-stage battery charging strategy. Fig. 3. Schematic of a two-stage battery charging strategy. 4.2 PIC microcontroller charging strategies for Li–iron batteries PIC series microcontrollers, including 8-, 16-, and 32-bit microcontrollers, are provided by Microchip Technology Inc.(5) As the controller core, a PIC18F8720 microcontroller(6) is employed with the following underlying key features: The way to charge a battery is found to significantly affect the service life of the battery. Hence, a recommended battery charge strategy can be found in an official guide provided by each battery manufacturer. Yet, it takes a relatively long time to charge a battery, namely, a slow paced charge strategy due to safety concerns. Nonetheless, high-performance charge strategies become a key issue in the development of battery chargers in an attempt to meet the cost reduction requirement in today’s highly competitive market. Batteries are characterized in official user guides in terms of cutoff voltage, charge voltage, maximum charge current, maximum continuous discharge current, and so forth. Hence, a high-performance battery charger must be developed in compliance with the specifications. As stated in Ref. 11, there are up to five ways to charge a Li–iron battery as a rule. The battery charging strategy employed herein consists of two stages, namely, the constant current mode followed by the constant voltage mode; such a strategy benefits from both charging modes. In this fashion, the charging time of a battery can be reduced considerably when operated in the constant current mode, while the service life of a battery can be well maintained when in the constant voltage mode. During the first stage of charging, the battery charger works in the constant current mode for efficiency concern. When the battery output voltage approaches a specified threshold, the charger switches to the constant voltage mode to protect the battery from overcharging. Charging continues until the voltage threshold is reached. Presented in Fig. 3 is an illustration of a two-stage battery charging approach. The first stage is the so-called bulk charging step, since high-speed battery charging is performed by a charger operating in the constant current mode. The process proceeds until a voltage threshold is reached. At this point in time, the battery is not fully charged, not in a 100% SOC. Hence, the charging process moves into the second stage, that is, the constant voltage mode. Over the second stage, the charge current falls steeply, but the battery voltage rises smoothly, with time, until another voltage threshold (a 100% SOC) is reached. 3696 Sensors and Materials, Vol. 31, No. 11 (2019) Fig. 3. 5.1 Step-down DC–DC converter In terms of circuit configuration, nonisolated DC–DC converters can be categorized into, for example, buck, boost, buck-boost, Ćuk, SEPIC, and Zeta converters.(12) A buck converter is employed herein owing to the advantage of easy hardware implementation. Sketched in Fig. 4 is the framework of a buck converter, also known as a voltage step-down converter, since the converter’s output voltage is kept below the input at all events. According to the volt-second balance, the input and output voltages are related,(12) Vo = VsD. (2) (2) (2) Vo = VsD. 5.3.2 Component choices Shown in Table 3 are the component choices for the proposed Li–iron battery charger.(12) LiFePO4 battery charger design 5.3 5.3.1 PV module specifications Table 2 gives the electric specifications of a Kyocera KC40T PV module,(13) according to which the presented battery charger is designed. As shown in Table 2, a Kyocera KC40T PV module provides an MPP output current of approximately 2.48 A. Hence, as many as 12 PV modules are cascaded as a high-speed battery charger to deliver a maximum output power of approximately 516 W toward an EA-480100-C15 LiFePO4 battery. The battery charger is designed to operate only when the output voltage VPV of the PV module array lies between 62 and 208 V with a maximum charge current iB of 10 A. The charging continues when the battery output voltage VB lies beyond 58 V and below 58.4 V. According to Eq. (2), the duty cycle D of the buck converter, illustrated in Fig. 5, is bounded by 0 21) ( 0 95) ( D . D D . max min = ≤ ≤ = (3) (3) 0 21) ( 0 95) ( D . D D . max min = ≤ ≤ = and the load resistance RL is given as and the load resistance RL is given as ( ) 58.4 5.84 10 VB RL iB = = = Ω. (4) (4) 5.2 Battery charger framework Demonstrated in Fig. 5 is a schematic diagram of the presented Li–iron battery charger mainly based on a buck converter with an inductor L1, a capacitor C1, a high-speed switching diode D1, and an IGBT transistor SW1. Through a skillful manipulation of the duty cycle D in the buck converter, an MPP is tracked and the PV module array serves as a battery charger. The output voltage of a voltage divider formed from the resistors R3 and R4 and the current sensed by a current sensor are fed into a microcontroller, and then the battery output voltage VB and current iB are evaluated through the built-in ADCs. According to the detected voltage, the battery charger is operated in the preset mode. When the charge current falls below a specified level owing to an insufficient supply of solar radiation, the MPP tracker is enabled for efficiency improvement. As the first step, a fraction of the PV module output voltage is fed into the microcontroller through a voltage divider consisting of the resistors R1 and R2, and then the PV module output voltage is found by means of a built-in ADC. Subsequently, an MPP tracking task is performed by regulating the PV module output voltage at the specified maximum power voltage on condition that the battery voltage lies below 58 V, as illustrated in Fig. 6. 3697 Sensors and Materials, Vol. 31, No. 11 (2019) Fig. 4. Framework of a buck converter. Fig. 5. (Color online) Framework of the presented battery charger controller. Fig. 4. Framework of a buck converter. Fig. 4. Framework of a buck converter. Fig. 4. Framework of a buck converter. Fig. 5. (Color online) Framework of the presented battery charger controller. Fig. 6. Control flow chart of the presented battery charger. Fig. 6. Control flow chart of the presented battery charger. 3698 Sensors and Materials, Vol. 31, No. 11 (2019) Sensors and Materials, Vol. 31, No. 11 (2019) LiFePO4 battery charger design 5.4 Measurements Presented in Fig. 7 is a photograph of the implemented charge controller, programmed in C language, with a PIC18F8720 chip as the core. In consideration of measurement efficiency improvement, a PVS0120 PV simulator(14) is employed to simulate the performance of an array of Kyocera KC40T PV modules, as specified in Table 2, and to conduct an MPPT performance test at various irradiation levels. The simulated PV module array offers an output voltage of 208 V, an output current of 2.48 A, and an output power of 516 W, and the irradiation level is adjustable by a rotary knob. Table 2 Electric specifications of KC40T PV module. Type: KC40T Maximum output power 43 W Current of maximum power point 2.48 A Voltage of maximum power point 17.4 V Open voltage 21.7 V Short current 2.65 A Table 3 Component choices for the employed buck converter. Part Specification and model Inductor 120 μH/AWG16# Capacitor 33 μF/100 V Semiconductor switch G4PH50UD/50 A, 1200 V Diode 40EPF06/40 A, 600 V Table 3 Component choices for the employed buck converter. Part Specification and model Inductor 120 μH/AWG16# Capacitor 33 μF/100 V Semiconductor switch G4PH50UD/50 A, 1200 V Diode 40EPF06/40 A, 600 V 3699 Sensors and Materials, Vol. 31, No. 11 (2019) Fig. 7. (Color online) Photograph of the implemented battery charger. Fig. 7. (Color online) Photograph of the implemented battery charger. Fig. 8. Well-regulated output voltage of a PV module through a MPPT in response to an abrupt change in the level of incident solar radiation. Fig. 9. The same experiment as in Fig. 8 is repeated except for a solar radiation change from 400 to 800 and then back to 400 W/m2. Fig. 9. The same experiment as in Fig. 8 is repeated except for a solar radiation change from 400 to 800 and then back to 400 W/m2. Fig. 9. The same experiment as in Fig. 8 is repeated except for a solar radiation change from 400 to 800 and then back to 400 W/m2. Fig. 8. Well-regulated output voltage of a PV module through a MPPT in response to an abrupt change in the level of incident solar radiation. Fig. 9. The same experiment as in Fig. 8 is repeated except for a solar radiation change from 400 to 800 and then back to 400 W/m2. 5.4.1 Measured MPPT performance Presented in Fig. 8 is the measured performance of a constant-voltage MPPT approach in response to an abrupt drop in the level of solar radiation from 1000 to 600 W/m2 at t = 15 s and then an abrupt rise back to 1000 W/m2. There is evidence that the output voltage of the PV module array is well regulated at a specified level by the presented MPP tracker against abrupt irradiation changes. The same experiment is repeated again for the MPPT performance test except that the irradiation level rises from 400 to 800 W/m2 and then plunges to 400 W/m2. As shown in Fig. 8, the output voltage of the PV module array in Fig. 9 is found to be well regulated again. 3700 Sensors and Materials, Vol. 31, No. 11 (2019) Sensors and Materials, Vol. 31, No. 11 (2019) 5.4.2 Measured battery charger performance Demonstrated in Fig. 10 are the measured PWM and charge current waveforms. In the presence of sufficient solar radiation, the charge current can be regulated at 10 A by the efficient manipulation of the PWM duty cycle via a PIC18F8720 chip. As can be found in Fig. 11, the battery charger continues to operate in the constant current mode until a voltage threshold of 58 V is reached. Subsequently, the battery charger works in the constant voltage mode, during which a charge current drop of approximately 0.01C amps occurs. 5.4.3 High-current charge/discharge characteristic For there is a nonuniform distribution of electrolyte in the interior of a Li–iron battery owing to redox reaction, particularly during a high current charge/discharge process, the battery cannot be measured until an equilibrium is reached. Illustrated in Fig. 12 is a flow chart for the Fig. 12. Flow chart of a battery charge/discharge cycle. Fig. 12. Flow chart of a battery charge/discharge cycle. Fig. 10. Measured PWM and charge cur rent waveforms in constant current mode. Fig. 11. Measured PWM and battery voltage waveforms in constant voltage mode. Fig. 10. Measured PWM and charge cur rent waveforms in constant current mode. Fig. 10. Measured PWM and charge cur rent waveforms in constant current mode. Fig. 11. Measured PWM and battery voltage waveforms in constant voltage mode. Fig. 11. Measured PWM and battery voltage waveforms in constant voltage mode. Fig. 11. Measured PWM and battery voltage waveforms in constant voltage mode. Fig. 12. Flow chart of a battery charge/discharge cycle. 3701 Sensors and Materials, Vol. 31, No. 11 (2019) measurement of the battery charge/discharge characteristic curves. A fully discharged battery reaches a 0% SOC and then remains idle for 1 h. A two-stage charging strategy is performed subsequently, that is, the battery is firstly charged at a constant current of 1C amp until the cutoff voltage is reached. The battery charger is then operated in the constant voltage mode until the charge current falls to 0.01C amps, a state treated as a 100% SOC. The battery remains idle for another hour, and then another battery discharge task is performed at a constant current of 1C amp. A TES-33 battery capacity tester,(15) a product launched by TES Electric Electronic Corp., Taiwan, is adopted to plot graphs of the battery voltage, current, and SOC to understand battery behaviors over charge/discharge cycles. Provided in Fig. 13 is a charging characteristic curve of an EA-480100-C15 Li–iron battery. It is found that the battery voltage rises rapidly at a constant charge current of 1C amp until a threshold of 53.29 V is reached, and then continues to increase linearly, but slowly, with time. In the constant current mode, the SOC rises linearly and rapidly with the battery voltage. 5.4.3 High-current charge/discharge characteristic (Color online) Plot of battery temperature versus time in the case in Fig. 13. Fig. 15. (Color online) Discharge characteristic curve for an EA-480100-C15 Li–iron battery at 1C amp. agreement with the specified 10 AH battery capacity. The battery voltage decreases linearly with time from the initial 52.01 to 48.51 V together with an SOC of 11.86% over a timespan of approximately 55 min. Subsequently, the battery voltage plunges to 45.63 V, at which voltage, SOC is considered to be 0% herein. An average voltage of 48.82 V is observed over the entire discharge process, indicating the high performance of the Li–iron battery. If the energy conversion efficiency of the battery is defined as the ratio of “(average discharge voltage) * (average discharge capacity)” to “(average charge voltage) * (average charge capacity)”, the measurement results show that the energy conversion efficiency of this energy storage system is 90%. This energy conversion efficiency is much higher than those of other charge/discharge controllers. 5.4.3 High-current charge/discharge characteristic At a 1C amp charge current, it takes as short as 45.7 min to reach an 80.2% SOC, and the charger operation is switched to the constant voltage mode once a voltage threshold of 58.4 V is reached. Furthermore, it takes 64.8 min to reach a 90.1% SOC. The charge current plunges once a cutoff voltage of 58.4 V is reached, and the battery charger is operated in the constant voltage mode. Hence, it is evident that the discharge timespan of an EA-480100-C15 Li–iron battery, as a solar energy storage device, can be reduced significantly. Presented in Fig. 14 is a plot of battery temperature against time at a charge current of 1C amp for an EA-480100-C15 Li–iron battery. In the constant current mode, the temperature rises with time from 28.9 to 31.4 °C until a voltage threshold is reached. Subsequently, the charger operation is switched to the constant voltage mode, and the battery temperature and particularly the charge current start to fall instantly. A temperature rise lower than 3 °C is observed in the battery charge process in Fig. 14, eliminating the risk of overheating. Provided in Fig. 15 is a measured discharge characteristic curve of an EA-480100-C15 Li–iron battery at a discharge current of 1C amp. As indicated in the figure, it takes the battery 59 min or so to reach the cutoff voltage together with a 4.4% SOC, a finding in good Fig. 13. (Color online) Two-stage charge characteristic curve for an EA-480100-C15 Li–iron battery at 1C amp in the constant current mode followed by the constant voltage mode. Fig. 13. (Color online) Two-stage charge characteristic curve for an EA-480100-C15 Li–iron battery at 1C amp in the constant current mode followed by the constant voltage mode. Fig. 13. (Color online) Two-stage charge characteristic curve for an EA-480100-C15 Li–iron battery at 1C amp in the constant current mode followed by the constant voltage mode. 3702 Sensors and Materials, Vol. 31, No. 11 (2019) Fig. 15. (Color online) Discharge characteristic curve for an EA-480100-C15 Li–iron battery at 1C amp. Fig. 15. (Color online) Discharge characteristic curve for an EA-480100-C15 Li–iron battery at 1C amp. Fig. 14. (Color online) Plot of battery temperature versus time in the case in Fig. 13. Fig. 14. (Color online) Plot of battery temperature versus time in the case in Fig. 13. Fig. 14. 6. Conclusions In this paper, we presented an MPPT-based high-performance Li–iron battery charger, based on a buck converter and implemented on a PV system. A constant voltage approach is employed to perform an MPP tracking task. Moreover, a two-stage battery charging strategy is adopted in this work, i.e., charging operation in the constant current mode during the first stage of charging and then in the constant voltage mode during the second stage. Consequently, a LiFePO4 battery is charged rapidly at 1C amp, while an MPP tracker is enabled so as to deliver the maximum power to the battery in the event that the charge current falls below 1C amp. In contrast, the MPP tracker is disabled when the charge current is beyond 1C amp. The battery charger continues to operate in the constant current mode until a voltage threshold of 58 V is reached. At this point in time, the charger starts charging and then continues to operate in the constant voltage mode until another voltage threshold of 58.4 V is reached. In this manner, the presented two-stage high-performance strategy is validated as an effective approach not merely to speed up battery charging, but also to protect batteries from overcharging by temperature monitoring. It is worth mentioning that this high-performance charge/discharge controller uses only low-cost voltage, current, and temperature sensors. 3703 Sensors and Materials, Vol. 31, No. 11 (2019) DeviceDoc/cn011897.pdf 7 T. Župa and O. Líška: IEEE 8th Int. Symp. Applied Machine Intelligence and Informatics (SAMI) (2010) 241–242. https://doi.org/10.1109/SAMI.2010.5423730 241–242. https://doi.org/10.1109/SAMI.2010.5423730 8 A. K. Padhi, K. S. Nanjundaswamy, and J. B Goodenough: J. Electrochem. 144 (1997) 1188. https://doi.or 10.1149/1.1837571 9 A. K. Padhi, K. S. Nanjundaswamy, C. Masquelier, S. Okada, and J. B. Goodenough: J. Electrochem. 144 (1997) 1609. https://doi.org/10.1149/1.1837649 10 C. Masquelier, A. K. Padhi, K. S. Nanjundaswamy, and J. B. Goodenough: Solid State Chem. 135 (1998) 22 https://doi.org/10.1006/jssc.1997.7629 1 D. I. Stroe, M. Swierczynski, A. I. Stroe, and S. K. Kær: Batteries 2 (2016) 1. https://doi.org/10.3390/ batteries2040037 12 S. C. Wang: Power Electronic, Tung Hua Booj Co. Ltd. (2001) ISBN13: 9789576369131.i : Power Electronic, Tung Hua Booj Co. Ltd. (2001) ISBN1 13 Kyocera Photovoltaic Module KC40T Specifications, Kyocera Solar Industries (May 2007). 14 PV Simulator-PVS0120 Specifications. http://www.kernel-sys.co.jp/product/PVsimulator.pdf 15 http://www.tes.com.tw/33c.htm 15 http://www.tes.com.tw/33c.htm Sensors and Materials, Vol. 31, No. 11 (2019) References 1 S. H. Yang and T. C. Tsai: Sci. Dev. 309 (2005) 50. 2 Y. J. Huang: Master of Thesis, National Changhua University of Education, Changhua, Taiwan (2006). 2 Y. J. Huang: Master of Thesis, National Changhua University of Education, Cha 2 Y. J. Huang: Master of Thesis, National Changhua U 2 Y. J. Huang: Master of Thesis, National Changhua University of Education, Changhua, Taiwan (2006). 3 N K i N M b db d R O tbib R bl S t i bl E R 68 (2017) 1 htt //d i 3 N. Karamia, N. Moubayedb, and R. Outbibc: Renewable Sustainable Energy Rev. 68 (2017) 1. https://d org/10.1016/j.rser.2016.09.132 4 O. Ezinwannea, F. Zhongwena, and L. Zhijunb: Energy Procedia 107 (2017) 297. wannea, F. Zhongwena, and L. Zhijunb: Energy Procedia 4 O. Ezinwannea, F. Zhongwena, and L. Zhijunb: Energ 5 http://www.microchip.com 6 “PIC18Fxx20 Data Sheet”, Microchip Technology Inc. (2003). http://ww1.microchip.com/downloads/c DeviceDoc/cn011897.pdf About the Authors Pi-Yun Chen received her Ph.D. degree in engineering science and technology from National Yunlin University of Science and Technology, in 2011. Now she is an associate professor in the Department of Electrical Engineering, National Chin-Yi University of Technology, Taiwan. Her current research interests include fuzzy systems and advanced control systems. Kuei-Hsiang Chao received his B.S. degree in electrical engineering from National Taiwan Institute of Technology, Taipei, Taiwan, in 1988, and his M.S. and Ph.D. degrees in Electrical Engineering from National Tsing Hua University, Hsinchu, Taiwan, in 1990 and 2000, respectively. He is presently a professor at the National Chin-Yi University of Technology, Taichung, Taiwan. His areas of interest are computer-based control systems, applications of control theory, renewable energy, and power electronics. He is a life member of the Solar Energy and New Energy Association and a member of the IEEE. Yu-Sheng Tsai received his M.S. degree in electrical engineering from National Chin-Yi University of Technology in 2012. He is presently an engineer in Tranmax Machinery Co., Ltd. His research interests include photovoltaic power systems and energy storage systems.
https://openalex.org/W2337912392	https://www.int-res.com/articles/esr2016/29/n029p179.pdf	English	null	Hawksbill nest site selection affects hatchling survival at a rookery in Antigua, West Indies	Endangered species research	2,015	cc-by	7,112	Hawksbill nest site selection affects hatchling survival at a rookery in Antigua, West Indies Megan Reising1, Michael Salmon1,, Seth Stapleton2,3 1Department of Biological Sciences, Florida Atlantic University, Boca Raton, Florida 33431, USA 2Jumby Bay Hawksbill Project, St. John’s, Long Island, Antigua, West Indies 3Department of Fisheries, Wildlife and Conservation Biology, University of Minnesota, St. Paul, Minnesota 55108, USA Megan Reising1, Michael Salmon1,, Seth Stapleton2,3 ABSTRACT: Nesting populations of Critically Endangered hawksbill sea turtles remain depleted across much of their range in the Caribbean. Some islands, however, including Jumby Bay (Pasture Bay) in Antigua, have shown a steady increase in the number of nesting females. Furthermore, in recent years nesting has occurred in particularly high densities within the remnant maritime forest on the northwestern side of the bay, concentrating the entry of emerging hatchlings into the sea along a small (~160 m long) length of shoreline. Previous studies have shown that when many hatchlings enter the sea from a restricted location, aquatic predators may exploit that site. We fol- lowed 49 hatchlings by kayak at night as they swam offshore, and we determined that predation rates were significantly higher on the western than on the eastern side of the bay. At both locations, the turtles showed no obvious differences in offshore orientation that might have increased their vulnerability to predators. We hypothesize that the greater predation rate was most likely caused by the presence of more predators. To reduce those predation pressures, we recommend a 2-pronged strategy: (1) ‘risk-spreading’ (releasing hatchlings at other locations adjacent to, and within, the bay), and (2) habitat restoration to expand the area of attractive nesting habitat. KEY WORDS: Hawksbill · Eretmochelys · Predation · Nest density · Management · Predator−prey interactions © The authors 2015. Open Access under Creative Commons by Attribution Licence. Use, distribution and reproduction are un - restricted. Authors and original publication must be credited. *Corresponding author: salmon@fau.edu ENDANGERED SPECIES RESEARCH Endang Species Res ENDANGERED SPECIES RESEARCH Endang Species Res Vol. 29: 179–187, 2015 doi: 10.3354/esr00708 Published online December 10 OPEN PEN ACCESS CCESS Publisher: Inter-Research · www.int-res.com Study site This study was completed between July and September 2012, at Pasture Bay, Long Island (here- after Jumby Bay), Antigua, West Indies (17° 09’ N, 61° 45’ W; Fig. 1). Pasture Bay is U-shaped and bor- dered by a ~650 m long beach that extends to 2 points: Pasture Point to the west and Homer Point to the east (Fig. 1). The beach is backed by low- lying vegetation dominated by 4 species: seagrape Coco lo ba uvifera, inkberry Scaevola sericea, coco - nut palm Cocos nucifera, and green buttonwood Conocarpus erectus. The far northwest side of the beach contains the last remnants of the original maritime forest. This hypothesis was reinforced by reports of what can happen when managers deliberately concentrate nests in an effort to protect them from terrestrial threats such as predators, artificial lighting or poach- ers (Stancyk 1982, Wyneken & Salmon 1994, An - drews et al. 2003). Managers transfer clutches of eggs to adjacent, safer beach sites where they are reburied, sometimes inside fenced or guarded enclo- sures. These ‘hatcheries’ may contain hundreds of nests reburied side by side on the same evening. At the end of incubation some 50 to 60 d later, hatch- lings can emerge from several nests on the same evening and enter the sea from the same location, seaward of the hatchery. Protection as a manage- ment objective breaks down if predators locate those areas, presumably through learning. When this hap- pens, the area in front of a hatchery can become a ‘feeding station’ where predators wait and where fewer turtles survive (Wyneken & Salmon 1994, Mor- timer 1999, Pilcher et al. 2000). We completed transects across the bay to quantify depth, to locate reefs that could provide shelter for potential predators, and to identify those predators that were most likely to take hatchlings swimming offshore (Stancyk 1982, Gyuris 1994, Pilcher et al. 2000, Stewart & Wyneken 2004). The outer transect was made by swimming slowly on a straight path between Pasture and Homer points. Observers used snorkeling gear and dove occasionally to the bottom to inspect coral patches. Depth (water sur- face to sand bottom) was measured from a kayak travelling on the same path using a weighted line that was marked at 1 m intervals. Measurements were made at 4 locations, spaced equally between the points and the center of the bay. INTRODUCTION However, protection has resulted in some popula- tion increases, particularly in parts of the Caribbean (e.g. Beggs et al. 2007, NMFS 2007). One such popu- lation is found in Pasture Bay, Antigua, where researchers began monitoring turtles via saturation tagging in 1987. This population has more than dou- bled from ~30 females in late the 1980s to ~70 in 2012, and the number of nests has increased from ~110 to more than 250 annually over the same period (Jumby Bay Hawksbill Project [JBHP] Annual Re - ports, at www. jbhp.org). This increase has been brought about not only by the return of experienced females, but also by the addition of new recruits rec- ognized by the absence of tags or tag scars. Cur- rently, over 200 nests each year are marked and, after the hatchlings emerge, over 100 nests are exca- Current estimates indicate a 90% decline in world- wide populations of the hawksbill turtle Eretmo - chelys imbricata in all major oceans over the last 100 yr (Mortimer & Donnelly 2008). The causes are well known. This species has been hunted for food, for its eggs, and for its strikingly mottled shell plates. Its feeding grounds in tropical coral reef habitats are threatened by pollution and climate change, and its nesting sites are being altered and compromised to support tourism and other varieties of coastal devel- opment. In spite of efforts over many years to con- serve and protect this species, their numbers in most locations continue to decline (Meylan & Donnelly 1999, Mortimer & Donnelly 2008). Publisher: Inter-Research · www.int-res.com Endang Species Res 29: 179–187, 2015 180 correlated with an increase in predation rates on hatchlings swimming offshore; the opposite (eastern) side essentially served as a control since fewer turtles nested there. Pasture Bay is ideal for such an assess- ment as it is small enough to quantify survival rates for swimming hatchlings released from different locations. We also investigated whether there might be alternative explanations for any observed differ- ences in predation rate based on when, seasonally, the nests were deposited or how accurately the hatchlings oriented toward deep water. Turtles that orient poorly are likely to spend more time in shallow water where they are more vulnerable to predators (Whelan & Wyneken 2007). Our results indicated that predation rates were significantly elevated on the western side of the bay. INTRODUCTION vated to determine clutch size and productivity (Richardson et al. 1999, 2006, Ditmer & Stapleton 2012). Hatching success (the proportion of the clutch that produces hatchlings) is high, averaging 78% (Ditmer & Stapleton 2012). Pasture Bay is a U-shaped cove, facing north-north- east. Historically, a maritime forest was located behind the beach but most of that habitat has been re- moved except for one portion along the shoreline on the northwestern side of the bay. Since 1987 when the JBHP began keeping records, the pattern of nest dis- tribution along the beach has varied as landscape conditions have changed (JBHP, unpubl. reports). That variation is apparently correlated with shifts in the distribution of beach sands and the formation of embankments due to tidal surges, storms and hurri- canes, and changes in the vegetation planted behind the beach. Over the years, most of the nesting has oc- curred within the remnant maritime forest (which represents <30% of the available nesting areas in side the Bay) and within a mixture of cultivated and native vegetation in an adjacent beach site facing north. Hawksbills, unlike most species of marine turtles, typ- ically nest under a vegetation canopy (Horrocks & Scott 1991, Pérez-Castañeda et al. 2007). Over the last 6 yr, hawksbills have placed 35 to >50% of their nests within the forest (JBHP Annual Reports 2007−2012). Our hypothesis was that such a concentration of nests, and ultimately of hatchlings from those nests entering the sea from a restricted area, attracted aquatic pred- ators and led to higher predation rates. Study site We made sim- ilar and parallel transects at 2 additional locations approximately 1/3 and 2/3 of the distance closer to the shore. In this study, our goal was to determine whether the concentration of nests in the maritime forest was Reising et al. Hawksbill hatchling survival at Jumby Bay 181 in lunar illumination, water clarity, tidal phase or cloud cover. Fig. 1. (A) Antigua in the northeastern Caribbean. (B) The island of Jumby Bay is located ~2 km to the north of the main island. (C) Pasture Bay (arrow) is a U- shaped cove that ends at Pasture Point to the west and Homer Point to the east p Each hatchling towed a ‘Wither- ington float’ that consisted of a 5 × 1 cm wide balsa wood rod, carved into a streamlined shape (Stewart & Wyne ken 2004, Whelan & Wyneken 2007). A short (2.4 cm long) cold-chemical glow stick was glued into a cavity on top of the float. A counterweight attached to the bottom of the float kept the glow stick facing upward so its glow was visible only from above the water. The float was tethered to the turtle by a ~1.5 m length of lightweight black thread that encir- cled the hatchling just behind the front flippers. In previous studies, this device failed to attract preda- tors and only slightly reduced hatchling swimming speed (Pilcher et al. 2000, Stewart & Wyneken 2004). It is unlikely that tracking via the float system compromised hatchling survival, as even unen- bered marine turtle hatchlings are incapable of mming faster than their aquatic predators. Floats d ~10 m behind the kayak for 0.5 h at night (as a mby Bay is ow) is a U- to the east Fig. 1. (A) Antigua in the northeastern Caribbean. (B) The island of Jumby Bay is located ~2 km to the north of the main island. (C) Pasture Bay (arrow) is a U- shaped cove that ends at Pasture Point to the west and Homer Point to the east Hatchling collection Hatchlings were collected during August and Sep- tember. We placed plastic coated wire screening around nests that had been incubating for ~55 d to contain the hatchlings after an emergence. We mon- itored screened nests at half-hour intervals between 17:00 and 03:00 h. When an emergence occurred we released all of the turtles with the exception of 2 hatchlings that were retained so they could be followed to measure predation rates as they swam offshore. Open Sand Reef Reef Seagrass Fig. 2. Aerial view of Pasture Bay, August 2012. Arrows: re- gions of reef near the shoreline, seagrass beds (primarily Thalassia testudinum) near the center shoreline, the open sand bottom in deeper water with clumps of coral inter- spersed, and in some instances rising above the water sur- face at low tide. Red rectangle: division of the shoreline into 2 approximately equal east and west halves. The remnant maritime forest is located on the northwest side of the bay Open Sand Reef Reef Seagrass Spatial pattern of nest placement A total of 211 nests were deposited in Pasture Bay between June and November, distributed as 62 nests in the eastern half and 149 nests in the western half. The number of nests in each half differed significantly from equivalence (105.5, χ2 = 17.8, p < 0.001, df = 1). Our null hypothesis was that there would be no dif- ference in predation rates on hatchlings released on the 2 sides of the bay. This hypothesis was rejected when χ2 test p-values were ≤0.05 (Siegel & Castillan 1988). Measuring predation rates Hatchlings were followed offshore by kayak to estimate predation rates. Trials took place within minutes after an emergence occurred (between 17:00 and 03:00 h) and were almost equally divided between those that were staged on the west and on the east side of the bay (Fig. 2). Up to 4 turtles were followed each night when weather permitted (light wind, small waves). One group of turtles was tracked on the east and the other on the west side of the bay. This procedure minimized the probability that pre- dation rates were influenced by nightly differences Fig. 2. Aerial view of Pasture Bay, August 2012. Arrows: re- gions of reef near the shoreline, seagrass beds (primarily Thalassia testudinum) near the center shoreline, the open sand bottom in deeper water with clumps of coral inter- spersed, and in some instances rising above the water sur- face at low tide. Red rectangle: division of the shoreline into 2 approximately equal east and west halves. The remnant maritime forest is located on the northwest side of the bay Fig. 2. Aerial view of Pasture Bay, August 2012. Arrows: re- gions of reef near the shoreline, seagrass beds (primarily Thalassia testudinum) near the center shoreline, the open sand bottom in deeper water with clumps of coral inter- spersed, and in some instances rising above the water sur- face at low tide. Red rectangle: division of the shoreline into 2 approximately equal east and west halves. The remnant maritime forest is located on the northwest side of the bay Fig. 2. Aerial view of Pasture Bay, August 2012. Arrows: re- gions of reef near the shoreline, seagrass beds (primarily Thalassia testudinum) near the center shoreline, the open sand bottom in deeper water with clumps of coral inter- spersed, and in some instances rising above the water sur- face at low tide. Red rectangle: division of the shoreline into 2 approximately equal east and west halves. The remnant maritime forest is located on the northwest side of the bay Endang Species Res 29: 179–187, 2015 182 Site characteristics Once fitted with the tether and float, each hatch- ling was allowed to crawl down the beach (with the float held in the air above and behind the turtle), enter the bay, and begin swimming. Turtles were fol- lowed by kayak at a distance of 5 to 10 m. A hand- held GPS (Garmin Geko 201TM, accuracy: ±3 m) was used to record hatchling location at 5 min inter- vals. Each turtle was followed until it either left the bay, was an estimated 400 m offshore, had been swimming for at least 30 min, or was taken by a pred- ator. Surviving hatchlings were recaptured, untied and released. Pasture Bay is deepest (≤4 m) at the center of the transect made farthest offshore between the bay’s 2 points. The bay becomes progressively shallower along the 2 parallel transects located 1/3 and 2/3 of the distance toward the shore (Fig. 2). Large patches of mostly dead coral border the shallows on either side of the bay; many smaller patches are scattered inside the bay, with the tops of some exposed during low tide. A limestone bed varying in width is present in the shallows near shore, and is covered with sea- grass (primarily Thalassia testudinum; Fig. 2) near the center of the shoreline. Hatchling fate (taken by a predator or survived its trial) was noted on a battery-powered voice recorder along with the turtle’s final GPS location, the approx- imate depth, a brief description of the bottom habitat, and the duration of the turtle’s swimming activity. If the turtle was taken by a predator, the float was usu- ally recovered nearby at the surface with the thread severed. We completed several daytime surveys in an effort to identify and count any fish (or other) predators that might take hatchlings, but none were seen. RESULTS test) were not attacked by predators, nor did any predators attack the float shortly before, during or after attacking a hatchling. Swimming speeds and offshore orientation We followed 49 hatchlings (25 hatchlings released from the west and 24 released from the east side of the bay) as they swam offshore (Fig. 3). On the west- ern side, 3 hatchlings survived (predation rate = 88%) whereas on the eastern side 18 hatchlings sur- vived (predation rate = 25%). Predation rates were significantly higher on the west side of the bay (Fisher exact test p < 0.05). Predated hatchlings were typically consumed too soon after release to accurately measure their swim- ming speed; speeds were therefore determined using data from the surviving hatchlings. Speeds (m min−1) were calculated by dividing the distance (m) each turtle had travelled by the time spent swimming. Val- ues were converted from m min−1 to the more typically used km h−1 to facilitate comparisons with other studies. Tracking was done when sea state conditions inside the bay were either calm (no wind) or when light winds (from the N, NE or SE) generated small waves (≤30 cm in height) that did not interfere with observations or kayak maneuverability. Tidal ampli- tudes in Antigua (≤30 cm) are small and were unlikely to affect predation rates. Hatchlings were most often taken by predators over reefs (n = 19 obs); less often they were taken over sand, seagrass or mixed sand/reef bottom profiles (n = 9 obs). Preda- tion sites varied in distance from the shoreline between 17 to 301 m, and in time from the onset of swimming between 2 min 58 s and 27 min. On aver- Hatchling offshore orientation was determined by the compass direction between the site where the turtle entered the water and its location when the trial ended (either by its release or by its disappear- ance after being taken by a predator). We used Oriana 3 (Kovach Computing Services) to calculate a group mean angle and dispersion (95% confi- dence limit) for the turtles released on each side of the bay. Rayleigh tests (Zar 1999) were used to deter- mine if the 2 groups of turtles preferred to swim in a generally similar direction (e.g. were significantly oriented). Reising et al. Hawksbill hatchling survival at Jumby Bay 183 0 2 4 6 8 10 0.31–0.50 0.51–0.70 0.71–0.90 0.91–1.10 Frequency (no. of hatchlings) Swimming speed (km h–1) Fig. 4. Distribution of swimming speeds shown by the 18 sur- viving hawksbill hatchlings released on the east side of Pas- ture Bay. Swimming speeds and offshore orientation Mean swimming speed of the 3 surviving turtles released from the west side of the Bay was 0.85 km h−1 0 2 4 6 8 10 0.31–0.50 0.51–0.70 0.71–0.90 0.91–1.10 Frequency (no. of hatchlings) Swimming speed (km h–1) A B Fig. 3. (A,B) Paths taken by hawksbill hatchlings as they swam offshore from (A) the west (n = 25) and (B) east (n = 24) side of Pasture Bay, shown separately for greater clarity. Black line in (B) divides the bay into approximately equal west and east halves. Red tracks represent hatchlings taken by predators. Circle diagrams in bottom panels show the ori- entation of the turtles in each group: north (0°), east (90°), south (180°), west (270°). Blue dots = single turtles. Arrows point to the group mean angle. Both groups are significantly orien ted (Ray leigh test p < 0.01). Photos: Google Earth A B Fig. 4. Distribution of swimming speeds shown by the 18 sur- viving hawksbill hatchlings released on the east side of Pas- ture Bay. Mean swimming speed of the 3 surviving turtles released from the west side of the Bay was 0.85 km h−1 Fig. 4. Distribution of swimming speeds shown by the 18 sur- viving hawksbill hatchlings released on the east side of Pas- ture Bay. Mean swimming speed of the 3 surviving turtles released from the west side of the Bay was 0.85 km h−1 Fig. 4. Distribution of swimming speeds shown by the 18 sur- viving hawksbill hatchlings released on the east side of Pas- ture Bay. Mean swimming speed of the 3 surviving turtles released from the west side of the Bay was 0.85 km h−1 Fig. 3. (A,B) Paths taken by hawksbill hatchlings as they swam offshore from (A) the west (n = 25) and (B) east (n = 24) side of Pasture Bay, shown separately for greater clarity. Black line in (B) divides the bay into approximately equal west and east halves. Red tracks represent hatchlings taken by predators. Circle diagrams in bottom panels show the ori- entation of the turtles in each group: north (0°), east (90°), south (180°), west (270°). Blue dots = single turtles. Arrows point to the group mean angle. Both groups are significantly orien ted (Ray leigh test p < 0.01). Photos: Google Earth Factors affecting predation rates Predators took most of the hatchlings released from the western side of the bay whereas the majority of the turtles released from the eastern side survived. Several variables could potentially explain these results. One possibility was that predation rates on the hatchlings differed because turtles on the western side oriented on an offshore course to exit the bay less accurately than the turtles on the eastern side of the bay. The swimming paths of hatchlings exposed to ar- tificial lighting, for example, show more dispersion from a heading directly offshore than those of hatch- lings swimming away from dark beaches (Withering- ton 1991). Under such circumstances, hatchlings spend more time in shallow water, increasing the probability that they will be detected from below by predators (Gyuris 1994, Wyneken & Salmon 1994, Whelan & Wyneken 2007). At Pasture Bay, those possibilities ap- peared remote for several reasons. First, the bay is so shallow that if predators were present, no path in an offshore direction would enable the turtles to evade detection (Fig. 2). Second, most of the hatchlings swimming offshore on both sides of the bay were well oriented (suggesting artificial lighting from homes on the east side of the bay had no major impact on their performance) and showed relatively little deviation from paths that would ultimately lead them out of the bay and toward deep water (Figs. 2 & 3). age, predation events occurred after the turtles had been swimming for slightly over 11 min. Predation sites varied in depth between 30 cm and 3.7 m, with an average depth of 1.7 m. Management implications At some locations in the Caribbean, the numbers of adult, subadult, and juvenile hawksbills seen on the foraging grounds are increasing (Puerto Rico, Florida, and the US Virgin Islands, NMFS 2007), as are the numbers of nesting females at some key in - dex sites where long-term data are available. Among these are Barbados, Buck Island Reef National Mon- ument, Mona Island, and Jumby Bay, Antigua (Beggs et al. 2007, Richardson et al. 1999, 2006, NMFS 2007). These encouraging results suggest that hawksbill populations can recover when adequately managed and protected. Additional observations suggest that factors other than swimming speed more importantly influenced the probabilities of hatchling survival. On the east side of the bay, 4 of the 6 turtles taken by predators were lost in relatively deep, more open water after swimming for a longer portion of their trial period. In contrast, most of the turtles taken by predators on the west side of the bay were lost soon after their trial began, and in relatively shallow water (Fig. 3). Those differences, again, suggest a stronger correlation be - tween hatchling fate and location than between hatchling fate and swimming speed. On their foraging grounds, Caribbean populations of hawksbills consist of genetically mixed stocks that differ in their mtDNA, and thus represent distinct matrilines (Bass 1999, Abreu & Leroux 2007, NMFS 2007, Leroux et al. 2012, Proietti et al. 2014). When the time comes to breed, females segregate and migrate with strong fidelity to specific regional nest- ing sites. The genetic stock nesting in Pasture Bay was originally identified by Bass (1999) as unique to rookeries located at Antigua and Barbuda. It thus qualifies as a unique matriline that should be main- tained to preserve the genetic diversity of hawksbill populations nesting in the Caribbean Sea. We conclude that the elevated predation rate on the west side of Pasture Bay was most likely a conse- quence of a concentration of hatchlings (both in time as well as in space) at a location where predators could capture more prey. Our observations do not permit us to say whether the predators were re - sponding to prey abundance; they may have favored the west side of the bay for other reasons. Swimming speeds and orientation Swimming speeds of 18 hatchlings that survived their trial on the east side of the bay averaged 12.4 m min−1 (range: 5.0−17.7 m min−1), or 0.74 km h−1 (Fig. 4). Speeds for the 3 turtles that survived on the west side of the bay averaged 14.1 m min−1 (range: 13.22−14.95 m min−1), or 0.85 km h−1. Hatchlings oriented in directions that would enable most of the turtles to exit the bay (Fig. 3). The mean angle (±SD) on the east side of the bay was slightly west of north (354° ± 10°), whereas on the west side it was northeast (21° ± 7°). Both groups showed nearly identical significant group orientation (Rayleigh test, east: Z = 22.8 ,west: Z = 20.4, p < 0.01; Fig. 3) but no overlap between their 95% confidence limits, indica- ting that the 2 distributions differed statistically. Endang Species Res 29: 179–187, 2015 184 A second possibility is that the hatchlings released on each side of the bay differed in their swimming speeds, which in turn somehow affected their vulner- ability. Although we lack the data to exclude this possibility (because so few turtles on the western side of the bay survived long enough to obtain a reliable swimming speed measurement), this also seems unlikely. The turtles often pulled the same floats. Given the number of turtles tested (n = 49) over a span of several weeks, it is also unlikely that the dis- tribution of swimming speeds differed by chance. At the same time, swimming speed could have been a minor factor that affected turtle survival on the west side of the bay. The 3 survivors on the west side swam at an average speed (0.85 km h−1) that was faster than the average speed of the surviving turtles on the east side (0.74 km h−1; Fig. 4). This leads us to hypothesize that faster movement through an area containing many predators may be important for achieving even a small increase in survival probabil- ity on the western side of the bay. This was not the case on the eastern side as both slower as well as faster swimming turtles survived (Fig. 4). At a hatchery site in southeastern Florida, USA, tarpon Megalops atlanticus, mangrove snapper Lutjanus griseus, yellowtail jack Caranx hippos and reef squid Sepioteuthis sepioidea were common predators of hatchling loggerhead sea turtles Caretta caretta. Swimming speeds and orientation As was the case at Jumby Bay in the present study, none of these predators were seen in the area during the day. However, all made an appearance in front of the hatchery after dusk where they consumed the turtles, often within min- utes after they entered the sea (Wyneken & Salmon 1994). Management implications Our data indicate that hatchling survival at Pasture Bay might be compromised, but the evidence that such a reduction in hatchling numbers has a serious impact remains uncertain. Some of the ambiguities center on the following issues: (1) Since this is the first study of its kind at Pasture Bay, we do not know whether the predation rates we witnessed in 2012 are typical of other years, and especially of those years when nests were differ- ently distributed among the 2 halves. Continued monitoring will be essential to firmly establish the relationship between nest distribution patterns, predator distribution, and predation rates upon the hatchlings. (2) We do not know if predation rates based upon tracks of single hatchlings are representative of those of hatchlings taken by predators while swimming off- shore as a group with their siblings. The latter is the more typical situation for most marine turtles, in - cluding hawksbills (Witzell 1983), since hatchlings emerge from their nests in one large or in several smaller groups. (4) Finally, and in spite of the predation rates we document in this study, there is presently no evi- dence for a decline in the nesting population at Pas- ture Bay (Richardson et al. 2006, JBHP Annual Report for 2012, JBHP unpubl. data); rather, the pop- ulation is thriving and has increase two- to three-fold over the past several decades. However, the most recent estimates indicate that hawksbills in both the Atlantic and Pacific Ocean basins reach sexual matu- rity in 17 to 22 yr (review: Avens & Snover 2013). Going back 22 yr, about 30 hawksbills were nesting in Pasture Bay. That number may have been insuffi- cient to attract as many predators to the bay and so predation pressures on the hatchlings may have his- torically been less than those we find today. If so, then the increase in the number of nesting females observed during the previous years may not be sus- tained in the future. g To date, all studies have quantified predation rates on single hatchlings as they are followed offshore (Witherington & Salmon 1992, Gyuris 1994, Pilcher et al. 2000, Stewart & Wyneken 2004, Whelan & Wyne - ken 2007, Harewood & Horrocks 2008). When those rates are relatively low (≤6%; e.g. Management implications We can, however, state that the greater abundance of prey on the west side of the bay occurred because a majority of the nests (109 of 211) were placed within the rem- nant maritime forest, and that this situation ulti- mately compromised the survival of the hatchlings. We hypothesize that as a result, more predators were attracted to that site, as has been reported to occur under similar conditions in shallow waters in front of hatcheries (Wyneken & Salmon 1994, Mortimer 1999, Pilcher et al. 2000). Interestingly, while the number of females nesting in Pasture Bay and at peripheral beaches on Jumby Bay has increased over the years (Richardson et al. 2006, JBHP Annual Reports 2009−2012), hawksbill nesting elsewhere in Antigua remains depleted (Fuller et al. 1992, Meylan 1999, M. Clovis-Fuller, Antigua Sea Turtle Project, pers. comm.). Unless those trends are reversed, the females nesting at Pas- ture Bay may represent the only source of new recruits to this matriline. Those circumstances sug- gest the need for a conservative management strat- egy, one that promotes an increase both in produc- Reising et al. Hawksbill hatchling survival at Jumby Bay 185 [egg and hatchlings stages] are critical to maintain recruitment to the older stages’ (Heppell et al. 2003, p. 287), and so a complete loss of all of the hatchlings departing from the bay will not sustain the popula- tion. At the same time, ‘…valid input values for Car- ibbean hawksbills are simply not yet available’ (Crouse 1999, p. 186). Our results suggest that the rookery at Pasture Bay remains productive, as nests placed on the eastern side of the bay may contribute disproportionately to the number of hatchlings that survive to exit the bay. Are those numbers adequate to compensate for the losses to predators we describe here? Do the larger clutches of Antigua hawksbills (mean: 144 eggs per nest; JBHP Annual Report for 2012) make this possible? The steady increase in nesting activity over the years at Jumby Bay is encouraging and suggests a positive answer. How- ever, it is unclear whether the benefits accrue only to sites on Jumby Bay; mainland (Antigua and Barbuda) monitoring suggests modest in creases in nesting dur- ing recent years as well (M. Clovis-Fuller, Antigua Sea Turtle Project, pers. comm.). tive adult nesting and in hatchling survival of this Critically Endangered species (Meylan & Donnelly 1999). Management implications Stewart & Wyneken 2004, Whelan & Wyneken 2007, Harewood & Horrocks 2008) predation rate estimates are likely to be reliable because few predators are present. In contrast, where predation rates are higher (e.g. Gyuris 1994, Pilcher et al. 2000, present study), groups of turtles swimming together might affect more predators (perhaps positively, negatively, or not at all) as a result of a ‘dilution effect’ (Cresswell & Quinn 2011) or a ‘confusion effect’ (Ioannou et al. 2008). Until appropriate experiments are done, the nature of such effects remains unknown. Predation rates may also depend upon the kinds of predators as these may differ in specific strategies used to detect and capture prey, as well as in the number of prey each predator is capable of consuming on a given evening (small squid probably take a single hatch- ling, whereas each tarpon can consume many turtles; Wyneken & Salmon 1994). In spite of these uncertainties, there is no question that preserving the Pasture Bay matriline is a pre- ferred option, and so an effort should be made to improve those prospects by increasing hatchling pro- duction at Pasture Bay. With that end in mind, we recommend that in addition to the monitoring proto- cols currently in place, a short-term strategy should include the transfer and release of hatchlings from some nests to other locations within the Bay, and to adjacent beach sites on the island where the turtles are known to nest. ‘Spreading the spatial risk’ is rec- ommended when managing hatcheries (Mortimer 1999); it is also a strategy that was proven effective in (3) Another uncertainty centers on what constitutes an ‘acceptable’ versus an ‘unacceptable’ loss of hatchlings to predators, from the perspective of pop- ulation recovery. We know that ‘…inputs from both (3) Another uncertainty centers on what constitutes an ‘acceptable’ versus an ‘unacceptable’ loss of hatchlings to predators, from the perspective of pop- ulation recovery. We know that ‘…inputs from both Endang Species Res 29: 179–187, 2015 186 LITERATURE CITED reducing predation rates at a Florida, USA, hatchery site (Wyneken & Salmon 1994). Risk-spreading is also promoted by modifying the habitat to make it more suitable for nesting, with the objective that the turtles will distribute their nests more evenly. Management implications Efforts to do so are ongoing at Jumby Bay and should be continued by restoring the vegetation canopy behind the beach and selectively thinning sites with invasive Scaevola sericea to create entry ‘corridors’ (i.e. gaps in the vegetation) for females searching for nesting sites. Abreu A, Leroux R (2007) Hawksbills genetics explained. SWoT Rep 3: 16 Andrews HV, Choudhury BC, Shanker K (2003) Sea turtle conservation: beach management and hatchery pro- grammes. GOI−UNDP Project Manual, Centre for Her- petology/Madras Crocodile Bank GTrust, Mamallapu- ram, Tamil Nadu petology/Madras Crocodile Bank GTrust, Mamallapu- ram, Tamil Nadu Avens L, Snover ML (2013) Age and age estimation in sea turtles. In: Wyneken J, Lohmann KJ, Musick JA (eds) The biology of sea turtles, Vol 3. CRC Press, Boca Raton, FL, p 97−134 Bass AL (1999) Genetic analysis to elucidate the natural his- tory and behavior of hawksbill turtles (Eretmochelys imbricata) in the wider Caribbean: a review and re- analysis. Chelonian Conserv Biol 3: 195−199 We also suggest initiating 2 new research projects. One project should aim to identify and determine the abundance of the hatchling predators and assess their habitat requirements, movements and activity patterns. That knowledge should prove invaluable in the development of strategies to control their impact. A second project should be designed to directly determine what proportion of the hatchlings from controlled releases of entire clutches actually sur- vives to exit the bay. That objective could be accom- plished by recaptures of swimming hatchlings min- utes later in a shallow net floating at the surface, and anchored across the opening of the bay. A similar technique is used to estimate the abundance of juve- nile marine turtles in other shallow water habitats (Ehrhart 1983). These data may also be used to deter- mine whether tracking single turtles (a less labor- intensive procedure) provides a reliable estimate of hatchling survival probabilities. Beggs JA, Horrocks JA, Krueger BH (2007) Increase in hawksbill sea turtle Eretmochelys imbricata nesting in Barbados, West Indies. Endang Species Res 3: 159−168 ® Cresswell W, Quinn JL (2011) Predicting the optimal prey group size from predator hunting behavior. J Anim Ecol 80: 310−319 ® Crouse DT (1999) Population modelling and implications for Caribbean hawksbill turtle management. Chelonian Conserv Biol 3: 185−188 Ditmer MA, Stapleton SP (2012) Factors affecting hatch suc- cess of hawksbill sea turtles on Long Island, Antigua, West Indies. Acknowledgements. We are grateful to the National Save- the-Sea-Turtle Foundation of Fort Lauderdale, Florida, USA, for financial support. The Jumby Bay Hawksbill Project, which is generously supported by the Jumby Bay Island Company, provided logistical support. We thank the Jumby Bay Resort for use of their kayaks to survey Pasture Bay and to follow swimming turtles offshore. This study served as a portion of a Master’s thesis for M.R. She thanks her commit- tee members (N. Dorn and J. Wyneken) for their advice, guidance and encouragement. Comments by S. Heppell, J. Wyneken, M. J. Saunders, Matthew Godfrey and several referees improved the manuscript. The study design was approved by the Florida Atlantic University IACUC Com- mittee (protocol A12-16) and by the Antigua Division of Fisheries. Management implications IUCN/SSC Marine Turtle Specialist Group Publ 4, p 175−178 Grobois FA, Donnelly M (eds) Research and manage- ment techniques for the conservation of sea turtles. IUCN/SSC Marine Turtle Specialist Group Publ 4, p 175−178 Siegel S, Castillan NJ (1988) Nonparametric statistics for the behavioral sciences. McGraw-Hill, New York, NY Mortimer JA, Donnelly M (2008) Eretmochelys imbricata. In: IUCN 2008. IUCN Red List of Threatened Species ver- sion 2010.4. www.iucnredlist.org Stancyk SE (1982) Non-human predators of sea turtles and their control. In: Bjorndal KA (ed) Biology and conserva- tion of sea turtles. Smithsonian Institution Press, Wash- ington, DC, p 19−38 NMFS (National Marine Fisheries Service) (2007) Hawksbill sea turtle (Eretmochelys imbricata) 5-year review: sum- mary and evaluation. NMFS, Silver Spring, MD, and US Fish and Wildlife Service, Jacksonville, FL Stewart KR, Wyneken J (2004) Predation risk to loggerhead hatchlings at a high-density nesting beach in southeast Florida. Bull Mar Sci 74: 325−335 Pérez-Castañeda R, Salum-Fares A, Defeo O (2007) Repro- ductive patterns of the hawksbill turtle Eretmochelys imbricata in sandy beaches of the Yucatan Peninsula. J Mar Biol Assoc UK 87: 815−824 ® ® Whelan CL, Wyneken J (2007) Estimating predation levels and site-specific survival of hatchling loggerhead sea - turtles (Caretta caretta) from South Florida beaches. Copeia 2007: 745−754 ® ® Pilcher NJ, Enderby S, Stringell T, Bateman L (2000) Near- shore turtle hatchling distribution and predation. In: Pilcher NJ, Ismail G (eds) Sea turtles of the Indo-Pacific: research, management, and conservation. Asean Aca- demic Press, London, p 151−166 Witherington BE (1991) Orientation of hatchling loggerhead sea turtles at sea off artificially lighted and dark beaches. J Exp Mar Biol Ecol 149: 1−11 ® ® Witherington BE, Salmon M (1992) Predation on loggerhead turtle hatchlings after entering the sea. J Herpetol 26: 226−228 ® ® Proietti MC, Reisser J, Mrines LF, Rodriguez-Zarate C and others (2014) Genetic structure and natal origins of immature hawksbill turtles (Eretmochelys imbricata) in Brazilian waters. PLoS ONE 9: e88746 ® ® Witzell WN (1983) Synopsis of biological data on the hawks- bill turtle Eretmochelys imbricata (Linnaeus, 1766). FAO Fish Synop 137, Miami, FL Richardson JI, Bell R, Richardson TH (1999) Population ecology and demographic implications drawn from an 11-year study of nesting hawksbill turtles, Eretmochelys imbricata, at Jumby Bay, Long Island, Antigua, West Indies. Submitted: January 5, 2015; Accepted: October 19, 2015 Proofs received from author(s): December 7, 2015 Editorial responsibility: Matthew Godfrey, Beaufort, North Carolina, USA Management implications PLoS ONE 7(7): e38472 ® Ehrhart LM (1983) Marine turtles of the Indian River Lagoon system. Fla Sci 46: 337−346 Fuller JE, Eckert KL, Richardson JI (1992) WIDECAST Sea Turtle Recovery Action Plan for Antigua and Barbuda. Eckert KL (ed) CEP Tech Rep 16. UNEP Caribbean Envi- ronment Programme, Kingston Gyuris E (1994) The rate of predation by fishes on hatchlings of the green turtle (Chelonia mydas). Coral Reefs 13: 137−144 ® In conclusion, our data indicate that a concentra- tion of nesting sea turtles may lead to circum- stances that compromise hatchling survival during offshore migration. Given those circumstances and the Critically Endangered status of hawksbills throughout the Caribbean, we recommend addi- tional management strategies to improve nesting habitat suitability and refine estimates of nesting beach productivity. Harewood A, Horrocks J (2008) Impacts of coastal develop- ment on hawksbill hatchling survival and swimming suc- cess during their initial offshore migration. Biol Conserv 141: 394−401 ® Heppell SS, Snover M, Crowder LB (2003) Sea turtle popu- lation ecology. In: Lutz P, Musick JA, Wyneken J (eds) The biology of sea turtles, Vol 2. CRC Press, Boca Raton, FL, p 275−306 Horrocks JA, Scott NM (1991) Nest site location and nest success in the hawksbill turtle Eretmochelys imbricata in Barbados, West Indies. Mar Ecol Prog Ser 69: 1−8 ® Ioannou CC, Tosh CR, Neville L, Krause J (2008) The confu- sion effect: from neural networks to reduced predation risk. Behav Ecol 19: 126−130 ® Leroux RA, Dutton PH, Abreu-Grobois FA and others (2012) Re-examination of population structure and phylogeog- raphy of hawksbill turtles in the Wider Caribbean using longer mtDNA sequences. J Hered 103: 806−820 ® Meylan AB (1999) Status of the hawksbill (Eretmochelys imbricata) in the Caribbean region. Chelonian Conserv Biol 3: 177−184 Meylan AB, Donnelly M (1999) Status justification for listing the hawksbill (Eretmochelys imbricata) as critically endangered in the 1996 IUCN Red List of Threatened Animals. Chelonian Conserv Biol 3: 200−224 Mortimer JA (1999) Reducing threats to eggs and hatch- lings: hatcheries. In: Eckert KL, Bjorndal KA, Abreu- Reising et al. Hawksbill hatchling survival at Jumby Bay 187 ging data reveal a significant increase in nesting hawks- bill sea turtles (Eretmochelys imbricata) on Long Island, Antigua. Anim Conserv 9: 302−307 Grobois FA, Donnelly M (eds) Research and manage- ment techniques for the conservation of sea turtles. Management implications Chelonian Conserv Biol 3: 244−250 Wyneken J, Salmon M (1994) Aquatic predation, fish densi- ties, and potential threats to sea turtle hatchlings leaving from open-beach hatcheries. Tech Rep 94-11, Broward County Board of Commissioners, Fort Lauderdale, FL Zar JH (1999) Biostatistical analysis. Prentice-Hall, Upper Saddle River, NJ Richardson JI, Hall DB, Mason PA, Andrews KM, Bjorkland R, Cai Y, Bell R (2006) Eighteen years of saturation tag- ® ® Zar JH (1999) Biostatistical analysis. Prentice-Hall, Upper Saddle River, NJ Richardson JI, Hall DB, Mason PA, Andrews KM, Bjorkland R, Cai Y, Bell R (2006) Eighteen years of saturation tag- ® ® Editorial responsibility: Matthew Godfrey, Beaufort, North Carolina, USA
https://openalex.org/W4229778575	https://www.scielo.br/j/ram/a/PtKSFmHZc8pfxqgNVTvS9Ch/?lang=pt&format=pdf	Portuguese	null	Apresentação	RAM. Revista de Administração Mackenzie	2,010	cc-by	243	APRESENTAÇÃO Caro leitor, Este número da RAM é especialmente dedicado ao tema diversidade e inclu- são nas organizações. Este número da RAM é especialmente dedicado ao tema diversidade e inclu- são nas organizações. Recentemente, realizou-se, no Brasil, o II Fórum Internacional de Diversi- dade e Inclusão, no qual foram debatidos estudos sobre diversidade e inclusão no ambiente organizacional. A continuidade das discussões se constituiu em alvo da edição especial temática da RAM. Conclamamos os pesquisadores da área a submeter seus mais recentes trabalhos para composição desta edição. Neste momento, a RAM se sente engrandecida pela oportunidade de apre- sentar à comunidade acadêmica parte da produção científica vinculada a temá- tica tão central para o campo de conhecimento de administração. São apresen- tados seis artigos que julgamos de grande interesse e valor para o nosso público leitor e para o desenvolvimento do setor. Certamente não podemos deixar de enfatizar que, para elaboração desta edi- ção, contamos com a colaboração e o apoio de seleto grupo de avaliadores e auto- res. Destaca-se a participação internacional, reunindo pesquisadores do Brasil, dos Estados Unidos e de Portugal. A partir da edição 11.4, daremos continuidade à apresentação dos artigos submetidos às edições ordinárias da RAM. Passemos à introdução, realizada pelos editores convidados Darcy Mitiko Mori Hanashiro (UPM-Br) e Cláudio Vaz Torres (UnB-Br), aos quais agrade- cemos a dedicação ao processo editorial de elaboração deste número de nossa revista. Desejamos a todos uma profícua leitura! Walter Bataglia Editor acadêmico
https://openalex.org/W3002494973	http://journal.unika.ac.id/index.php/sisforma/article/download/2156/pdf	English	null	Designing Web-Based DISC Psychology Personality Analysis Tests	SISFORMA/Sisforma	2,020	cc-by	3,827	Designing Web-Based DISC Psychology Personality Analysis Tests Designing Web-Based DISC Psychology Personality Analysis Tests signing Web-Based DISC Psychology Personality Analysis Designing Web-Based DISC Psychology Personality Analysis Tests Daniel Aditama Somatdie Information System Departement, Soegijapranata Catholic University danieladitama11@gmail.com Erdhi Widyarto Information System Departement, Soegijapranata Catholic University erdhi@unika.ac.id Alb. Dwiyoga Widiantoro Information System Departement, Soegijapranata Catholic University yoga@unika.ac.id by order and harmonization [4]. To find an individual personality, the individual can take the personality psychology test. Personality psychology tests are professional psychology proficiency which include administration, assessment, and interpretation of the support of calculating traits and styles[5]. Abstract— Using a manual system in the companies or organizations today, has many disadvantages like inefficient, wasting time, require a lot of costs, and make work difficult. By integrating a DISC personality analytical psychological test with information system, the tests are carried out with computers initially using paper, so it more economical for using less paper. More than that, the test results are done automatically check by the computer, no need to manually check by the psychologist. In addition to being effective in examining, the validation of examinations is very high because it can recover human errors. g y One of the personality psychology tests is the DISC test. DISC theory is a psychological theory used to determine people's personalities based on people's emotional behavior based on their environment. DISC is divided into four personalities, there are: Dominance (D), Influence (I), Steadiness (S), and Compliance (C) [6]. The psychological test of DISC personality analysis is done on paper. Individuals who will be tested will answer on paper and then processed manually and calculated manually. But this system is less efficient and takes a lot of time. Keywords— online test, phycology test, DISC assessment online test, information system, computerized based test With the internet technology, which one is web-base, communication and information services can be done easily and efficiently [7]. To use technology in an organization must have an information system. An information system in an organization provides processes and information that is useful for members and clients of the organization. With this information system, they can operate more effectively [8]. Moreover, the content validity of technology-based test examinations is higher too. Content validity is a benchmark for reflecting the content domain as expected [9]. With technology, the goal is to reduce human error in examination, so the test results SISFORMA: Journal of Information Systems (e-Journal)Vol. 6 \| No. 2 \|Th. 2019. ISSN 2442-7888 (online) DOI 10.24167/Sisforma II. LITERATUR REVIEW E. Online DISC Personality Analysis Test The web-based DISC personality analysis psychological test application is used to classify personality types based on test results. In an application test experiment to select KKL participants at Amikom Purwokerto, from the experiment, 59% of the 6 questions through the questionnaire were approved by the respondents [17]. G. Waterfall Method Waterfall method is a method used for learning in developing a software [20]. First, the waterfall method was published by Walter Royce in 1970 [21]. The method of work of this method is that the conditions of the ongoing stage must be completed before proceeding to the next stage [22]. F. Hypertext Preprocessor (PHP) Hypertext Preprocessor (PHP) is a simple and powerful programming language designed to create HTML content. PHP can be run on many operating systems such as Linux, Solaris, FreeBSD, MacOS X and Windows. In addition, various web servers can be used, such as Apache, Microsoft IIS, and Netscape [18]. PHP can be obtained free of charge. To download PHP just by visiting the site www.php.net and applying it to the PHP editor [19]. B. Phycological Test Psychological test is a tool that are used as benchmarks for psychological aspects of the individual. Psychological test is containing interview questions that are packaged in written form [2]. I. INTRODUCTION Psychology test or psychological test is a tool used to find out a person's condition [1]. This psychological test is used to measure the functions of cognitive and emotional abilities such as intelligence, interests and talents, to the personality of the person [2]. The psychological test method is a psychology research tool that is important for the ability to be tested, for the example is attitudes, talents, personality traits, and interests [3]. Personality is all kinds of varied behaviors carried out by individuals who are regulated 82 Designing Web-Based DISC Psychology Personality Analysis Tests more valid. Utilization of computerized technology for DISC personality analysis psychological test from previously done paper-based test is an interesting part to be explored. Because, with the many advantages of using computers for psychological tests of DISC personality analysis can develop the organization and improve work effectiveness. C. DISC Theory y DISC theory was first put forward by William Moulton Marston, namely the expression of emotional behavior divided into four types of individuals when interacting with their environment: Dominance (D), Influence (I), Steadiness (S), and Compliance (C). Marston calls that four "primary emotions"[11]. DISC personality analysis test has been used by more than 50 million test users and published in books that have been translated in 35 languages [12]. Personality- based DISC tests are used to classify potential members into a group, and measure productivity from members. Beside that it is also useful for psychometric measurement, leadership, and activeness in the organization [13]. Although valid, the DISC test can change because the emotional experience of an individual tends to change rather than be eternal [14]. D. Computer Assisted Test Computer Assisted Test (CAT) is a test performed with a computer. Computers can be used in various uses as test media. Starting from the simplest, which is used to display exam questions, check test results, and give test scores[15]. So, the efficiency and validity of the answer check will increase [16]. A. Test According to Freidenberg in 1995, test is a type of assessment that had specific procedures for sending information and converting information to numerical forms. The Personality Analysis Test is a Typical performance test, a test used to measure typical performance. This test is usually used for typical behavior, thinking habits, and feelings[10]. SISFORMA: Journal of Information Systems (e-Journal)Vol. 6 \| No. 2 \|Th. 2019. ISSN 2442-7888 (online) DOI 10.24167/Sisforma III. RESEARCH METHODOLOGY The application development process used in this study uses the waterfall model. The application development began with collecting data through interviews with the Center for Applied Psychology (PPT) at Unika Soegijapranata. In addition to interviews, data searching is also done by looking for sources in books, journals, and 83 SISFORMA: Journal of Information Systems (e-Journal)Vol. 6 \| No. 2 \|Th. 2019. ISSN 2442-7888 (online) DOI 10.24167/Sisforma Designing Web-Based DISC Psychology Personality Analysis Tests Figure 1. Application Workflow other media that can be related to research. Continued for making application concepts. Making application concepts includes application flow, mockup, database design, and determining user roles. Continued with, making the program. The programming is done with the programming language PHP and Bootstrap Framework using the Visual Studio Code editor. The database is created using phpMyAdmin on a MySQL basis. After the program is finished, the application is tested. If the application is appropriate, the application will be implemented to the client and perform maintenance. After that, the application will be used as a research medium. In this study, the object of research was people, with the population being psychologists, staff, and participants from the Applied Psychology Unika Soegijapranata and samples used by 30 students. The source of the primary data of this study came from questionnaires given to 30 respondents and interviews with psychologists and PPT staff and secondary data sources of this study came from journals, articles, and literature regarding the psychological test of DISC. SISFORMA: Journal of Information Systems (e-Journal)Vol. 6 \| No. 2 \|Th. 2019. ISSN 2442-7888 (online) DOI 10.24167/Sisforma III. RESULTS AND DISCUSSION The results of this application design are called online DISC personality analysis psychological tests. This application consists of 3 user roles, namely participants, psychologists, and staff. Figure 1 is the flow diagram of the application. Figure 1. Application Workflow Figure 2. Application Use Case The use case of the DISC personality analysis psychological test application is as Figure 2. Designing databases using Entity Relational Diagrams (ERD) from DISC personality analysis psychological test applications is as Figure 3. Figure 2. Application Use Case 84 SISFORMA: Journal of Information Systems (e-Journal)Vol. 6 \| No. 2 \|Th. 2019. ISSN 2442-7888 (online) DOI 10.24167/Sisforma Designing Web-Based DISC Psychology Personality Analysis Tests Figure 3. Application ERD Figure 4. Display of Participant Question. Figure 3. Application ERD Figure 4. Display of Participant Question. Figure 6. Display of DISC Graph Figure 7. Display of Participant Conclusions Staff role is to add new users. Staff can also see user management. User management is used to view active users, edit user data, and delete users. Figure 6. Display of DISC Graph Figure 7. Display of Participant Conclusions Figure 3. Application ERD Figure 6. Display of DISC Graph Figure 6. Display of DISC Graph Figure 6. Display of DISC Graph Figure 7. Display of Participant Conclusions Figure 6. Display of DISC Graph Figure 7. Display of Participant Conclusions Figure 3. Application ERD Figure 3. Application ERD Figure 7. Display of Participant Conclusions Staff role is to add new users. Staff can also see user management. User management is used to view active users, edit user data, and delete users. Figure 8. Display Registering Participants Figure 9. Display User Management he questionnaire consists of 2 sessions, the espondents' demographic questions and Figure 8. Display Registering Participants Figure 4. Display of Participant Question. Figure 4. Display of Participant Question. Psychologists have the task of giving conclusions to answers the results that have been processed by the computer. The computer processing results are calculating the accumulated answers of participants, calculating the graph automatically, and showing conclusions of the behavior and work field that is suitable for participants. Figure 8. Display Registering Participants Figure 8. Display Registering Participants Figure 8. Display Registering Participants Figure 9. Display User Management Figure 5. Display of Answer Accumulation Figure 9. Display User Management Figure 9. Display User Management The questionnaire consists of 2 sessions, the respondents' demographic questions and application review questions Figure 5. Display of Answer Accumulation 85 SISFORMA: Journal of Information Systems (e-Journal)Vol. 6 \| No. 2 \|Th. 2019. ISSN 2442-7888 (online) DOI 10.24167/Sisforma 85 Designing Web-Based DISC Psychology Personality Analysis Tests A. Demographics of Respondents Figure 10. Age of Respondents A. Demographics of Respondents Figure 10. Age of Respondents A. Demographics of Respondents A. Demographics of Respondents As many as 86.67% of the 30 respondents had never done DISC personality analysis psychological tests. While respondents who had taken the DISC personality analysis test were 13.33%. Figure 10. Age of Respondents Figure 10. Age of Respondents Figure 14. Application Usage Understanding Diagram Figure 14. Application Usage Understanding Diagram Figure 10. Age of Respondents From 30 respondents, the majority of respondents were 20 and 21 years old as much as 40%. While respondents aged 19, 22, and 24 years were 6.67%. Figure 14. Application Usage Understanding Diagram Figure 11. Respondent Gender Diagram There are 16 respondents or 53.33% agree that understand how to use the application quickly. While 8 people or 26.67% answered strongly agree, 4 people or 13.33% answered that they did not understand quickly using the application and 2 people or 6.67% were neutral. Figure 11. Respondent Gender Diagram Figure 11. Respondent Gender Diagram Figure 15. Convenience and Activity Diagrams Application Login From 30 respondents, balanced sex data was taken. That is 50% male and 50% female. Figure 12. Respondents' Educational Diagram Figure 15. Convenience and Activity Diagrams Application Login Figure 12. Respondents' Educational Diagram A total of 20 respondents or 66.67% agreed that the login process was easy and smooth. While as many as 8 respondents or 26.67% strongly agree if the login process is smooth and easy and 2 respondents or 6.67% disagree if the login process is smooth and easy. Education taken by respondents at this time, as many as 93.33% of the 30 respondents were pursuing a degree. While 6.67% were taking high school. Figure 13. Ever Using Respondent's Application Diagram SISFORMA: Journal of Information Systems (e-Journal)Vol. 6 \| No. 2 \|Th. 2019. 8 ISSN 2442-7888 (online) DOI 10.24167/Sisforma Figure 13. Ever Using Respondent's Application Diagram g p y Figure 16. Smooth Start Application Application Button Diagram Figure 13. Ever Using Respondent's Application Diagram Figure 13. Ever Using Respondent's Application Diagram Figure 16. Smooth Start Application Application Button Diagram Figure 16. Smooth Start Application Application Button Diagram SISFORMA: Journal of Information Systems (e-Journal)Vol. 6 \| No. 2 \|Th. 2019. ISSN 2442-7888 (online) DOI 10.24167/Sisforma SISFORMA: Journal of Information Systems (e-Journal)Vol. 6 \| No. 2 \|Th. 2019. ISSN 2442-7888 (online) DOI 10.24167/Sisforma 86 Designing Web-Based DISC Psychology Personality Analysis Tests Both respondents who agreed and strongly agreed to have a total of 13 respondents or 43.33%. A. Demographics of Respondents While those who disagree are only 1 person or 3.33% and 3 more or 10% are neutral. 12 respondents or 40% were neutral that the warning when not answering the question was very helpful. The remaining 8 respondents or 26.67% agree, 5 respondents or 16.67% strongly agree, 4 respondents or 13.33% disagree and 1 respondent strongly disagrees or as much as 3.33%. 12 respondents or 40% were neutral that the warning when not answering the question was very helpful. The remaining 8 respondents or 26.67% agree, 5 respondents or 16.67% strongly agree, 4 respondents or 13.33% disagree and 1 respondent strongly disagrees or as much as 3.33%. Figure 17. Ease Diagram for Using the Start Button in the Application Problem Figure 20. Convenience Diagrams in the process of transferring applications Figure 17. Ease Diagram for Using the Start Button in the Application Problem Figure 20. Convenience Diagrams in the process of transferring applications A total of 15 respondents or as many as 50% agreed that the button starts in the matter of easy application to use. While 7 respondents or 23.33% felt strongly agree, 5 respondents or 16.67% Neutral, and 3 respondents or 10% did not agree. Respondents who agreed that the process of moving the application questions quickly and accurately was 18 respondents or 60%. While 7 respondents or 23.33% strongly agreed. A total of 4 respondents or 13.33% were neutral, and 1 respondent or 3.33% did not agree Figure 18. Easy to read fonts in question view Figure 21. Application Drawing Diagram Figure 18. Easy to read fonts in question view Figure 21. Application Drawing Diagram Respondents who agreed to feel easy to read the font in the display question as many as 18 respondents or 60%. While 8 respondents or 26.67% strongly agreed, 3 respondents or 10% were neutral and 1 respondent or as much as 3.33% did not agree. There are 13 respondents or 43.33% agree that the application system is interesting. While 11 respondents or 36.67% strongly agreed and 6 respondents or 20% were neutral. Figure 19. The Convenient Diagram answers the question with the warning feature SISFORMA: Journal of Information Systems (e-Journal)Vol. 6 \| No. 2 \|Th. 2019. 87 ISSN 2442-7888 (online) DOI 10.24167/Sisforma Figure 19. The Convenient Diagram answers the question with the warning feature Figure 22. Application Usage Chart is easier than conventional Figure 22. A. Demographics of Respondents Application Usage Chart is easier than conventional Figure 22. Application Usage Chart is easier than conventional Figure 19. The Convenient Diagram answers the question with the warning feature Figure 22. Application Usage Chart is easier than conventional Figure 22. Application Usage Chart is easier than conventional SISFORMA: Journal of Information Systems (e-Journal)Vol. 6 \| No. 2 \|Th. 2019. ISSN 2442-7888 (online) DOI 10.24167/Sisforma 87 SISFORMA: Journal of Information Systems (e-Journal)Vol. 6 \| No. 2 \|Th. 2019. ISSN 2442-7888 (online) DOI 10.24167/Sisforma Designing Web-Based DISC Psychology Personality Analysis Tests Twelve respondents or 40% were neutral in feeling that using the application was more convenient than the written psychological test. While as many as 6 respondents or 20% agreed, 7 respondents or 23.33% strongly agreed, and 5 people or 16.67% did not agree. 15 respondents or 50% agreed if the instruction display was interesting. While 7 respondents or 23.33% were neutral, 6 respondents or 16.67% strongly agreed, and 3 respondents or 10% did not agree. Figure 26. Comfort Diagram Layout of instruction display Figure 26. Comfort Diagram Layout of instruction display Figure 23 Convenience Diagrams in the process of transferring applications Figure 26. Comfort Diagram Layout of instruction display Both respondents agreed and neutral each amounted to 10 respondents or as much as 33.33%. While as many as 8 respondents or 26.67% strongly agree, and 2 respondents or 6.67% disagree. Figure 23 Convenience Diagrams in the process of transferring applications In attracting the login screen, 18 people or 60% agreed. While 9 respondents or 30% were neutral and 3 people or 10% strongly agreed. In attracting the login screen, 18 people or 60% agreed. While 9 respondents or 30% were neutral and 3 people or 10% strongly agreed. Figure 27. Comfort diagram in reading the instruction display font Figure 27. Comfort diagram in reading the instruction display font Figure 24 Comfort Diagram Layout login View Figure 27. Comfort diagram in reading the instruction display font Sejumlah 18 responden atau 60% setuju bahwa font di tampilan instruksi jelas dan mudah dibaca. Sementara 8 responden atau 26,67% sangat setuju, 2 responden atau 6,67% netral dan masing-masing 1 responden atau 3,33% tidak setuju dan sangat tidak setuju. Figure 24 Comfort Diagram Layout login View A total of 25 respondents or 83.33% agreed that the layout of the login display was appropriate and appropriate. A. Demographics of Respondents While 3 respondents or 10% were neutral and 2 respondents or 6.67% strongly agreed. Figure 28. Drawn diagram of the display in the application Figure 28. Drawn diagram of the display in the application SISFORMA: Journal of Information Systems (e-Journal)Vol. 6 \| No. 2 \|Th. 2019. 88 ISSN 2442-7888 (online) DOI 10.24167/Sisforma Figure 25. Drawn diagram of instruction display Figure 28. Drawn diagram of the display in the application Figure 25. Drawn diagram of instruction display Figure 28. Drawn diagram of the display in the application Figure 28. Drawn diagram of the display in the application Figure 25. Drawn diagram of instruction display Figure 25. Drawn diagram of instruction display SISFORMA: Journal of Information Systems (e-Journal)Vol. 6 \| No. 2 \|Th. 2019. ISSN 2442-7888 (online) DOI 10.24167/Sisforma 88 Designing Web-Based DISC Psychology Personality Analysis Tests Respondents who feel neutral if the display of interesting questions as many as 12 respondents or 40%. Respondents who agreed were 10 respondents or 33.33% and strongly agreed with 8 respondents or 26.67%. Respondents who feel neutral if the display of interesting questions as many as 12 respondents or 40%. Respondents who agreed were 10 respondents or 33.33% and strongly agreed with 8 respondents or 26.67%. 11 respondents or 36.67% suggested applications to others to use the application. While 10 respondents or 33.33% strongly recommend, 6 respondents or 20% neutral and 3 respondents or 10% do not recommend. Figure 29. The layout comfort of the application questions Figure 29. The layout comfort o application questions REFERENCES [1] C. Suwartono, “Alat Tes Psikologi Konteks Indonesia: Tantangan Psikologi di Era MEA,” J. Psikol. Ulayat, vol. 3, no. 1, p. 1, 2017. [1] C. Suwartono, “Alat Tes Psikologi Konteks Indonesia: Tantangan Psikologi di Era MEA,” J. Psikol. Ulayat, vol. 3, no. 1, p. 1, 2017. [2] I. M. Kabinani, S. Rostianingsih, and L. P. Dewi, “KARIR DAN TES MINAT JABATAN PADA PUSAT KARIR UNIVERSITAS.” Figure 29. The layout comfort of the application questions [3] Y. Jahja, Psikologi Perkembangan, Pertama. Jakarta: Kharima Putra Utama, 2011. A total of 18 respondents or 60% agreed that the layout of the display about the application is comfortable to see. While each 6 or 20% of respondents were very agree and neutral. [4] C. S. Hall and G. Lindzey, Psikologi Kepribadian 1 TEORI-TEORI PSIKODINAMIK (KLINIS). Yogyakarta: Kanisius, 1993. Figure 30. The smoothness of the next button to move the application problem [5] [5] American Psychological Association (APA), “Personality Assessment.” [Online]. Available: https://www.apa.org/ed/graduate/speci alize/personality. [Accessed: 26-Jun- 2019]. [6] [6] C. K. E. Goni, H. Opod, and L. David, “Gambaran kepribadian berdasarkan tes Disc mahasiswa Fakultas Kedokteran Universitas Sam Ratulangi Manado semester 1 tahun 2016,” vol. 4, 2016. Figure 30. The smoothness of the next button to move the application problem A total of 17 respondents or 56.67% agreed if the next button for switching about the application went well and smoothly. While 8 respondents or 26.67% strongly agreed, 3 respondents or 10% were neutral and each 1 respondent or 3.33% did not agree and strongly disagree. [7] K. Hernawati, “Evaluasi dan Penilaian Interaktif Berbasis Web,” 2006. [8] D. Avison and G. Fitzgerald, Information System Development, 4th ed. Singapore: McGraw-Hill Education (UK), 2006. Figure 31. Intention Diagram Suggesting Applications to Others [9] I. W. Santyasa, “ANALISIS BUTIR DAN KONSISTENSI INTERNAL TES,” pp. 1–12, 2005. [10] Supratiknya, Penilaian Hasil Belajar dengan Teknik Nontes, vol. 28, no. 12. 2006. [11] Wiley, “Research Report for Adaptive Testing Assessment,” John Wiley Sons, Inc., 2018. [11] Wiley, “Research Report for Adaptive Testing Assessment,” John Wiley Sons, Inc., 2018. Figure 31. Intention Diagram Suggesting Applications to Others Figure 31. Intention Diagram Suggesting Applications to Others SISFORMA: Journal of Information Systems (e-Journal)Vol. 6 \| No. 2 \|Th. 2019. ISSN 2442-7888 (online) DOI 10.24167/Sisforma 89 SISFORMA: Journal of Information Systems (e-Journal)Vol. 6 \| No. 2 \|Th. 2019. ISSN 2442-7888 (online) DOI 10.24167/Sisforma Designing Web-Based DISC Psychology Personality Analysis Tests [12] C. S. REFERENCES Jones and N. T. Hartley, “Comparing Correlations Between Four-Quadrant And Five-Factor Personality Assessments,” Am. J. Bus. Educ., vol. 6, no. 4, p. 459, 2016. [18] B. K. Rasmus Lerdorf, Kevin Tatroe, Programming PHP, First Edit. Sebastopol: O’Reilly Media, Inc., 2002. [19] L. Atkinson, Core PHP Programming, Second Edi. New Jersey, 2001. [13] C. A. Bhardwaj, M. Mishra, and S. Hemalatha, “An Automated Compatibility Prediction Engine using DISC Theory Based Classification and Neural Networks,” arXiv Prepr. arXiv1709.00539, 2017. [20] M. G. Limaye, Software Testing, First Repr. New Delhi: Tata Mc-Graw Hill, 2009. [21] K. Petersen, C. Wohlin, and D. Baca, “The Waterfall Model in Large-Scale,” Prod. Softw. Process Improv. 10th Int. Conf. PROFES 2009, pp. 386–400, 2009. [14] I. Publishing, “DiSC® Classic and Models of Personality,” 1996. [15] F. Putrawansyah, “Aplikasi Computer Assisted Test (CAT) Pada Penerimaan Mahasiswa Baru Sekolah Tinggi Teknologi Pagar Alam (STTP),” vol. 1, no. 1, pp. 1–8, 2017. [22] S. Balaji and M. Sundararajan Murugaiyan, “Waterfall Vs V-Model Vs Agile: A Comparative Study On SDLC,” Int. J. Inf. Technol. Bus. Manag., vol. 292, no. 1, pp. 26–30, 2012. [16] G. Lippey and F. B. Baker, Computer- assisted Test Construction. New Jersey: Educational Technology, 1974. [17] I. Santiko, A. Prasetyo, and P. N. Sukisno, “Implementation of Psychotest System Application Selection of KKL STMIK Participants in Purwokerto Amikom Using DISC ( Dominance-Influence-Steadiness- Compliance ),” vol. 4, no. 1, 2019. 90 SISFORMA: Journal of Information Systems (e-Journal)Vol. 6 \| No. 2 \|Th. 2019. ISSN 2442-7888 (online) DOI 10.24167/Sisforma
https://openalex.org/W4220931961	https://zenodo.org/records/6782779/files/s41598-022-08187-z-2.pdf	English	null	Congenitally blind adults can learn to identify face-shapes via auditory sensory substitution and successfully generalize some of the learned features	Scientific reports	2,022	cc-by	12,792	www.nature.com/scientificreports www.nature.com/scientificreports Roni Arbel1,3, Benedetta Heimler1,2,4 & Amir Amedi1,2 Roni Arbel1,3, Benedetta Heimler1,2,4 & Amir Amedi1,2 Unlike sighted individuals, congenitally blind individuals have little to no experience with face shapes. Instead, they rely on non-shape cues, such as voices, to perform character identification. The extent to which face-shape perception can be learned in adulthood via a different sensory modality (i.e., not vision) remains poorly explored. We used a visual-to-auditory Sensory Substitution Device (SSD) that enables conversion of visual images to the auditory modality while preserving their visual characteristics. Expert SSD users were systematically taught to identify cartoon faces via audition. Following a tailored training program lasting ~ 12 h, congenitally blind participants successfully identified six trained faces with high accuracy. Furthermore, they effectively generalized their identification to the untrained, inverted orientation of the learned faces. Finally, after completing the extensive 12-h training program, participants learned six new faces within 2 additional hours of training, suggesting internalization of face-identification processes. Our results document for the first time that facial features can be processed through audition, even in the absence of visual experience across the lifespan. Overall, these findings have important implications for both non-visual object recognition and visual rehabilitation practices and prompt the study of the neural processes underlying auditory face perception in the absence of vision. Faces represent a common category of visual objects, and identification of their exemplars is the most prevalent object-identification task performed by sighted individuals1. Among other visual categories of objects, including man-made objects, words, and numbers, faces are unique, as their identification is performed almost exclusively through the visual modality. For example, man-made objects are easily accessible and therefore also identifiable via tactile manipulation in sighted and in blind individuals2, and reading is accessible via the tactile modality using the Braille code3. Another factor separating faces from other categories of visual objects is that expertise in face identification continues to evolve through the first decade of life4,5, with evidence of different processes mediating face identification in different developmental stages and clinical conditions. Specifically, in young children and those with face-identification impairments, face identification is based on featural processing, with a gradual development of adult-like holistic face processing at around school age6–8. This change in the perceptual process underlying face perception is accompanied by the development of a neural network for face processing, including regions in the visual cortex presenting cortical selectivity to faces over other visual stimuli4. Scientific Reports \| (2022) 12:4330 www.nature.com/scientificreports/ mainly due to social conventions. Consequently, congenitally blind adults are exposed to facial shapes less than their sighted peers. Thus, through tactile exploration blind people might learn the main perceptual charac- teristics of faces, but they might develop a limited ability to differentiate among face exemplars—if any at all. Indeed, face-shapes are a quite homogenous category of objects and humans need extensive exposure to a lot of different exemplars to reach an accurate face-discrimination ability. Second, unlike visual perception, during tactile perception each face part is explored separately, therefore further limiting the ability to construct holistic face representations9 which might facilitate identification processes. Both of the aforementioned reasons may therefore prevent the mental creation of a complete face representation in congenitally blind adults, potentially limiting face-shape perception in this population9.i g p p p p p Here we investigate for the first time the extent to which congenitally blind people can learn to perceive, identify and therefore accurately discriminate among face-shapes during adulthood via an atypical sensory modality. To this end, we transformed visual faces into audition, using an in-built visual-to-auditory trans- formation algorithm (i.e., sensory substitution). We chose audition because it is a sensory modality offering relatively higher resolution in frequency and time than touch10, therefore potentially allowing to convey richer face-related information. In addition, audition is not typically used for shape-based object recognition, including face-shapes, in everyday life, therefore excluding that resulting learning may be facilitated by previous experience and training. Specifically, visual-to-auditory Sensory Substitution Devices (SSDs) translate information carried via vision into audition using in-built algorithms that preserve the exact shape and location of features, creating what we call soundscapes, the interpretation of which can be learned by users via specific training11,12. Previous studies conducted on congenitally blind adults demonstrated the efficacy of visual-to-auditory SSD for various object-recognition tasks. Yet because of the need to down-sample each image to optimize SSD transformations, and given the lack of SSD training programs for advanced users, previous SSD studies on auditory shape recogni- tion focused on simple images11,13. Full faces were excluded because these complex visual images contain many details that must be correctly perceived to allow identification of specific exemplars, making it an extremely difficult SSD training—if possible at all. www.nature.com/scientificreports/ With the current study, we therefore also aimed to test the limits of SSD learning via audition, i.e., the extent to which it is possible to learn to perceive complex visual objects through sound, ultimately also starting to address the open issue regarding the extent to which current SSDs can be used as rehabilitation tools for everyday life conditions14. We reasoned that our in-built visual-to-auditory SSD algorithm, called the EyeMusic11,12 might facilitate the learning of complex soundscapes due to the fact that it embeds the transformation into sound of a unique visual dimension lacking in other visual-to-auditory SSDs, namely, color (see below and see “Methods” for details on the algorithm’s transformation rules). Indeed, color is known to enhance discriminability of images, especially in lower resolutions, and has been directly shown to enhance real-life face identification in the context of low visual resolution15. While it is difficult to directly project on the blind population the same color-related benefits, we reasoned that the EyeMusic color-transformation may facilitate the differentiation among auditory face features, i.e., similarly to what color does for hard visual tasks (see also16 showing the benefits of adding the EyeMusic color feature to the reading of words composed of letters from a newly learned SSD alphabet).t y p In greater detail, the EyeMusic is based on the sweep-line technique in which an image is scanned from left to right; each pixel of the image is translated into a musical note based on its location in the image, as well as its color. Specifically, pixels located lower on the image are translated into sounds of a lower musical note, while pixels located high on the image are translated into sounds of a higher musical note. In addition, positioning in the x-axis is conveyed via time, such that portions of the image located more to the left will be heard first. Five colors are represented by five musical instruments (red is mapped to an organ note, green to a reed’s tones, blue to brass instruments, yellow to string instruments, and white to voices of a choir; black is represented by silence).i , y g , ; p y ) We developed a specific methodology to train a group of congenitally blind adults, who have had already 50–70 h of past experience with SSDs, to identify complicated multi-colored images of cartoon faces. Roni Arbel1,3, Benedetta Heimler1,2,4 & Amir Amedi1,2 g g p g y In sum, while sighted individuals consistently engage the vast cognitive, perceptual, and neural resources required to perform face identification, congenitally blind individuals represent a unique group, excluded from the common experience of face-shapes since birth.hif p p This raises the question of whether accurate identification of different face exemplars based only on their shape-features can arise in blind people. Indeed, although the blind population can, in principle, perceive face shapes via touch, tactile face exploration has several crucial limitations. First, it does not happen very often, 1Department of Medical Neurobiology, Hebrew University of Jerusalem, Hadassah Ein-Kerem, Jerusalem, Israel. 2The Institute for Brain, Mind and Technology, Ivcher School of Psychology, Reichman University, Herzeliya, Israel. 3Department of Pediatrics, Hadassah Mount Scopus Hospital, Jerusalem, Israel. 4Center of Advanced Technologies in Rehabilitation (CATR), The Chaim Sheba Medical Center, Tel Hashomer, Israel. email: roni.arbel@ mail.huji.ac.il \| https://doi.org/10.1038/s41598-022-08187-z Scientific Reports \| (2022) 12:4330 www.nature.com/scientificreports/ www.nature.com/scientificreports/ First, to engage participants in the challenging training program, lasting ~ 12 h, and to promote efficient learning17,18, we inserted the training within the ecological situation of the “Guess Who?” children’s game. Participants were familiar with the game, yet until now had no accessible way to participate in. Notably, despite their previous SSD experience, soundscapes of face-shapes were for our participants a behavioral jump compared to previously trained soundscapes, as briefly stated also above. Indeed, face-shape soundscapes included multi-colored filled visual images that when transformed into audition, required the analysis of numerous sounds simultaneously (see19 for a discussion of the challenges in discriminating features on the Y axis in sweep-line based SSDs). Second, in order to facilitate and simplify the learning of these complex face-shape soundscapes and to allow participants to familiarize with the resulting complex sounds, we developed the strip-method to be used during the early stages of the training program, i.e., before introducing the full soundscapes. Specifically, we divided each visual face in three horizontal strips (upper, middle and lower face parts) and presented to participants one strip at a time (see “Methods” for further details and see Fig. 2 and “Supplementary material” for all our soundscapes). At the end of the program, each participant was tested on several tasks including face-shape identification. In addition, we tested whether face-shape identification could extend to the (untrained) inverted orientation of the same faces. We reasoned this was a particularly hard task as the inverted images created entirely different soundscapes from those of the trained faces. In addition, to perform identification of the inverted faces, par- ticipants were supposed to manipulate the inverted stimulus (e.g., mentally rotate it) to perform a correct face identification. Therefore, with this task, we aimed at testing the extent to which our participants were able to manipulate the learned face properties. Lastly, we tested face-learning dynamics, asking whether the principles learned during the ~ 12-h intense training program could be quickly applied to a novel set of cartoon faces. To this aim, we trained participants to perceive and learn to identify a novel set of 6 cartoon faces in 2 h of additional training, and compared this rapid learning to the learning of the first cohort of faces. https://doi.org/10.1038/s41598-022-08187-z Scientific Reports \| (2022) 12:4330 \| www.nature.com/scientificreports/ Figure 1. Training congenitally blind adults’ auditory identification of cartoon faces. www.nature.com/scientificreports/ Eye-Music transformation algorithm: Each image is scanned from left-to-right using a sweep-line approach so that the x-axis is mapped to time (i.e., characters positioned more on the left of the image are heard first). After downsampling the image to the EyeMusic’s resolution (50 × 30 pixels) the y-axis is mapped to the frequency domain using the pentatonic scale, such that parts of a character which appear higher in the image will be sonified with a higher pitch. Color is mapped to musical instruments. Red, white, blue, yellow, and green are transformed into organ notes, choir, brass instruments, string instruments, and reed tones, respectively. (A) An example of a real face transformed via the EyeMusic algorithm. (B) An example of a trained (cartoon) image transformed via the EyeMusic algorithm. Figure 1. Training congenitally blind adults’ auditory identification of cartoon faces. Eye-Music transformation algorithm: Each image is scanned from left-to-right using a sweep-line approach so that the x-axis is mapped to time (i.e., characters positioned more on the left of the image are heard first). After downsampling the image to the EyeMusic’s resolution (50 × 30 pixels) the y-axis is mapped to the frequency domain using the pentatonic scale, such that parts of a character which appear higher in the image will be sonified with a higher pitch. Color is mapped to musical instruments. Red, white, blue, yellow, and green are transformed into organ notes, choir, brass instruments, string instruments, and reed tones, respectively. (A) An example of a real face transformed via the EyeMusic algorithm. (B) An example of a trained (cartoon) image transformed via the EyeMusic algorithm. Figure 2. Experimental paradigm. The experiment was divided into three parts. First, participants underwent six hours of training, followed by interim testing of face identification and detection of changes to facial features (1b). The strip-method was used in this first part: each face was divided into three horizontal strips representing the upper, middle, and lower parts of the face (1a). To teach participants the composition of face parts and how to tune their hearing to perceive multiple tones at the same time, they were trained to perceived details within each strip. Following completion of this stage, participants were able to integrate their knowledge and perceive the complete face soundscapes. Second, participants underwent additional 6 h of whole-face training (2a). They subsequently took part in several tasks designed to test face identification, including the identification of learned faces in the untrained, inverted orientation (2b). Third, participants underwent an additional pre-post experiment, in which they quickly learned a new cohort of six faces and were tested on face identification before and after two hours of training (3a–3c). the new color of the changed feature. Verbal responses were registered. Participants were able to identify the change in 70.24% of the soundscapes (SD = ± 15.85%, chance level 50%), to localize it to the correct feature/loca- tion with an accuracy of 52.38% (SD = ± 7.93%, chance level 20%), and to identify the new color with 43.45% (SD = ± 9.58%, chance level 20%) accuracy (Fig. 3B). the new color of the changed feature. Verbal responses were registered. Participants were able to identify the change in 70.24% of the soundscapes (SD = ± 15.85%, chance level 50%), to localize it to the correct feature/loca- tion with an accuracy of 52.38% (SD = ± 7.93%, chance level 20%), and to identify the new color with 43.45% (SD = ± 9.58%, chance level 20%) accuracy (Fig. 3B). Part 2: Face identification tests following 12 h of training (n = 9). Experiment 1: Face identifica‑ tion. In this task, participants were presented with a face-shape soundscape and asked to identify it (i.e., by tell- ing his/her name). Results showed that following ~ 12 h of dedicated face-shapes training, participants were able to identify face-shapes with 87.38% (± 7.96%) accuracy, significantly above chance level (16.67%, t(8) = 26.53, p < 0.001, d = 8.76) (Fig. 3C). Results P t 1 I Part 1: Interim exploratory testing of face training progress (after 6 h of training). Assessment 1: guess who game: yes/no questions on facial features and naming task (n = 7). To explore participants ability in extracting shape-features among the soundscapes following the first stage of face-training, we asked them yes/no questions regarding the faces, and registered their verbal responses. Questions were directed towards face-features, such as “does this character wear glasses?”, “is this character bearded?”. Results showed partici- pants correctly answered questions with 72.77% accuracy (standard deviation—SD = ± 5.33%, chance level 50%) (Fig. 3A). When separating face features into visually large details (such as hair, beard, etc.) and visually small details (glasses, eyes, etc.), participants were able to identify large visual features with 93.41% accuracy (SD = ± 5.31%) and small features with 58.65% accuracy (SD = ± 8.28%) (Fig. 1A).t After responding to all feature-related questions for each face, participants were asked to name the face (once per face in this first assessment). Participants were able to name faces with 66.67% accuracy (SD = ± 21.52%, chance level 16.7% Fig. 1A). Assessment 2: Detection of change in facial features (n = 7). In addition to asking simple yes/no questions on the face-shapes soundscapes, we further assessed participants’ ability to extract image details by embedding changes in the features’ colors within the trained faces. For instance, changes included a change in hair color, glasses color, eye color (see Fig. 2 for some examples). We asked participants whether they could detect the change, and then to localize it to a specific facial feature within the image (hair, glasses, etc.), and finally, to identify https://doi.org/10.1038/s41598-022-08187-z Scientific Reports \| (2022) 12:4330 \| www.nature.com/scientificreports/ Figure 2. Experimental paradigm. The experiment was divided into three parts. First, participants underwent six hours of training, followed by interim testing of face identification and detection of changes to facial features (1b). The strip-method was used in this first part: each face was divided into three horizontal strips representing the upper, middle, and lower parts of the face (1a). To teach participants the composition of face parts and how to tune their hearing to perceive multiple tones at the same time, they were trained to perceived details within each strip. Following completion of this stage, participants were able to integrate their knowledge and perceive the complete face soundscapes. Second, participants underwent additional 6 h of whole-face training (2a). Results P t 1 I They subsequently took part in several tasks designed to test face identification, including the identification of learned faces in the untrained, inverted orientation (2b). Third, participants underwent an additional pre-post experiment, in which they quickly learned a new cohort of six faces and were tested on face identification before and after two hours of training (3a–3c). Discussion h k In this work, we show that congenitally blind adults could successfully identify colorful face-shapes exemplars via the auditory modality, despite life-long minimal experience with such shapes, following a tailored and engaging ~ 12-h training program with a visual-to-auditory Sensory Substitution Device (SSD). Moreover, we show that blind individuals were not only able to identify SSD-presented trained face-shapes, but quite strikingly, could also generalize face-shape identification to the same faces presented in their untrained, inverted orientation. The identification of face images in their inverted orientation requires the perception of a sound with completely different spectral properties than those of the trained, upright corresponding exemplar, potentially paired with the mental rotation of the face-shape representation. Finally, we show that the comprehensive 12-h training program with the original cohort of six faces enabled participants to rapidly learn (i.e., after 2 additional hours of dedicated training) a new set of six face-shapes. The colorful images of faces used in our study represent the most complicated category of images systematically trained using SSD to this point. This work has implications in the fields of visual and auditory processing, learning dynamics, mental imagery, and rotation, as well as practical implications for rehabilitation of blind and visually impaired/restored individuals, as discussed below. Face‑shape identification via the auditory modality. In this work we show that despite the lack of life-long extensive experience with face-shapes, congenitally blind adults can learn to perceive such shapes via the auditory modality and coherently organize this auditory perception to allow the identification of face-shapes exemplars. p To enable the learning of face images via auditory SSD, we developed a tailored training program that aimed at maximizing the learning of visual spatial face-feature configuration. Specifically, we avoided teaching facial features separately, as can be done through touch. Rather, we presented our face stimuli using the strip-method; for each face-shape, we created three horizontal strips, one for each of the uppermost, middle, and lowermost sections of the face (Fig. 2). Then, to promote participants’ engagement with complete face exemplars, during training, participants were encouraged to discuss facial features and their spatial arrangement within the faces. Arguably, the use of this spatial configuration approach to face-shape learning may have allowed participants to better integrate the face parts and build a “mental image” of the complete face object, and this, in turn, may have facilitated recognition of individual exemplars20. www.nature.com/scientificreports/ p = 0.006, d = 1.09). Finally, participants were significantly more accurate at identifying upright than inverted characters (t(16) = 4.78, p < 0.001, d = 1.59) (Fig. 3E). p = 0.006, d = 1.09). Finally, participants were significantly more accurate at identifying upright than inverted characters (t(16) = 4.78, p < 0.001, d = 1.59) (Fig. 3E). Part 3: Face identification of six additional characters after 2 additional hours of training (n = 9). Experiment 4: Discrimination of first cohort vs. second cohort of trained faces before and after 2 h of additional face training. Before and after the additional 2-h training, participants were presented with both co- horts of faces and were asked to discriminate the soundscapes as either belonging to the first (previously trained) or second cohort (newly trained) of learned faces. Participants provided their answer via response keys. Before training, participants successfully discriminated trained from novel faces with 80.2% (SD ± 5.5%) accuracy. A t-test against chance (50%) confirmed performance was above chance level (t(8) = 15.55, p < 0.001, d = 5.50), indicating the auditory properties of the novel faces were distinctive enough to enable their discrimination from the previously trained cohort of faces (Fig. 3F).t p y ( g ) When we repeated the same experiment after training, we observed a trend of improved discrimination between the two cohorts of faces; success rate was 85.4% (SD ± 15.4%), significantly above chance level as well (50%, w = 36, p = 0.014). However, comparing performance before and after training revealed no significant training effect (t(8) = 1.14, p < 0.29, d = 0.40). Namely, discrimination performance did not significantly improve following training. Similarly, participants were not significantly faster in discriminating previously learned faces from the cohort of newly-trained characters presented during the 2 h of additional training. Discrimination took, on average, 4.16 s (SD ± 1.23 s) before training and 4.04 s (SD ± 1.02 s) after training (t(14) = 0.56, p = 0.30, d = 0.20) (Fig. 3F). Experiment 5: Identification of six additional characters trained for 2 h. In this task, participants were presented with the set of newly trained face-shapes and asked to identify all exemplars via verbal responses. www.nature.com/scientificreports/ Results showed participants could successfully identify all recently learned faces, correctly identifying 96.74% (SD ± 3.36%) of the faces, on average, significantly above chance level (16.67%) (t(7) = 74.45, p < 0.001, d = 25.71). Participants were not only accurate; they were also quick in identification, and did so in 3.52 s, on average (SD ± 1.33 s) (Fig. 3G).i ( g ) Finally, to investigate learning dynamics, we compared the accuracy rate of the identification of previously learned faces (from Experiment 1) with that of novel faces (from the current experiment) using a paired-samples Wilcoxon signed-rank test (see “Methods”). This test revealed a significant effect (w = 36, p = 0.014)), whereby participants were more accurate in identifying novel faces learned only for 2 h, than they were for the first set of learned faces (i.e., learned within 12 h of training). In addition to being accurate, participants were also signifi- cantly faster in identifying these newly trained faces compared to the previously learned set of faces (t(14) = 4.06, p = 0.005, d = 1.43) (Fig. 3G). When analyzing reaction times, we observed participants identified a face in 5.44 s, on average (about two face presentations; SD = ± 2.08 s) (Fig. 3C). Experiment 2: Upright vs. inverted (untrained) orientation discrimination. In this task, participants were asked to discriminate between the soundscapes of upright trained faces and soundscapes of the same faces presented in the inverted (untrained) orientation. They provided their answer via response keys. Results showed par- ticipants could correctly classify the presented face-shape’s orientation as either upright or inverted, in 86.56% (SD ± 11.54%) of the trials. A t-test against chance level (50%) confirmed they could classify a face’s orientation significantly above chance level (t(8) = 9.50, p < 0.001, d = 3.05). When analyzing reaction times, we observed par- ticipants classified face orientation in 4.40 s, on average (about two face presentations; SD = ± 1.14 s) (Fig. 3D). Experiment 3: Identification of upright and inverted faces. In this task participants were requested to identify face-shapes presented in both upright and in the inverted orientation, and provide their answer via response keys. Our results confirmed that participants were able to identify faces in the upright orientation, with an aver- age accuracy of 80.32% (standard deviation (SD) ± 8.7%), significantly above chance level (30%, t(8) = 16.28, p < 0.001, d = 5.44). They were also able to identify characters when presented in the untrained, inverted orienta- tion, with an average accuracy of 51.39% (SD ± 16.86%), again significantly above chance level (30%, t(8) = 3.27, https://doi.org/10.1038/s41598-022-08187-z Scientific Reports \| (2022) 12:4330 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ In addition, both before and after training, they managed to provide an answer within 2 soundscape’s repetition (right bar graph). G. Following the 2-h training session, participants were successful at identifying newly-trained faces with higher accuracy than chance. They were significantly more accurate in the identification of newly compared to previously learned faces -accuracy level taken from Experiment 1 (left). In addition, they were also significantly faster in providing an answer when identifying newly compared to previously learned faces (right). In figures (A–G), error bars represent standard deviation. In figures (C–G), *p < 0.005, while p < 0.05. ▸ face-shapes recognition activate regions within the visual neural face recognition system22,23. Future studies should explore whether the case of face-shape recognition via the novel and non-ecological stimuli provided by the auditory soundscapes, tackles on object related, or rather, face related modules in the deprived visual cortex. bl d ll d d d l h h ft h f f l face-shapes recognition activate regions within the visual neural face recognition system22,23. Future studies should explore whether the case of face-shape recognition via the novel and non-ecological stimuli provided by the auditory soundscapes, tackles on object related, or rather, face related modules in the deprived visual cortex.t y p j p Notably, studies on visually restored individuals show that even after the restoration of functional vision, neural tuning to faces remains impaired2. For instance, although following cataract removal surgery, children can discriminate face from non-face exemplars24, a study on congenital cataract reversal surgery in children, showed the absence of the distinguished N170 marker for visual faces25. Functional Magnetic Resonance Imaging (fMRI) studies corroborate electrophysiological findings by showing individuals’ lack of functional selectivity to faces following prolonged visual deprivation26–29. Together, these results suggest that visual restoration may not be sufficient to establish accurate face-shapes recognition in sight-restored individuals. However, our results may encourage the hypothesis that SSD targeted rehabilitation, using dedicated and specific training, might be introduced after sight restoration to aid visual perception ultimately maximizing restoration outcomes30,31.i t g p p y g Moreover, following the completion of the training program, our participants were significantly quicker when learning to identify new exemplars belonging to the same group of objects (i.e., face-shapes), indicating that the perceptual concepts learned during training could be quickly exploited to learn novel stimuli. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Auditory face identification by congenitally blind adults. Part I: Interim exploratory testing of face training progress (after 6 h of training): (A) participants were requested to answer yes/no questions on the trained faces regarding facial features (e.g., does this character have blue eyes?) to test their ability to extract meaningful information regarding facial features. Results show that participants could correctly identify facial features, especially larger features. In addition, participants were also asked to name once each trained character, and were successful in doing so. (B) To further assure participants’ were able to detect colorful features within the complicated soundscapes, we changed some features of the trained faces (e.g., eye-colors, hair color etc.). Congenitally blind participants were relatively accurate to detect the presence of a change, but were less accurate when asked to localize the change (i.e., to identify the modified face feature) and to identify the changed color. Part II: Face identification tests following 12 h of training: (C) Participants were tested on face-shape identification of the 6 trained characters. Results show that they were able to correctly identify face- shapes with high accuracy, which was significantly higher than chance level (left bar graph). In addition, they were able to provide an answer within 2 soundscape’ repetitions (right bar graph). (D) Participants were able to successfully discriminate upright (trained) from inverted (untrained) faces with an accuracy significantly above chance level (left bar graph). In addition, we also show that it took them less than 2 soundscape’s repetition to provide an answer (right bar graph). E. Participants were asked to identify face-shapes in their upright (trained) and inverted (untrained) orientation, which resulted in a completely different soundscape. Results show that participants were able to identify untrained inverted faces with an accuracy that was significantly higher than chance level, but significantly lower than their accuracy in identifying the faces when presented in the upright and trained orientation. Part III: Face identification of a new cohort of 6 characters after 2 h of additional training: (F) Participants were requested to differentiate the previously trained faces from those belonging to the new cohort, before and after a 2-h training session. Results show that participants were able to do it with an accuracy significantly above chance level, both before and after the 2-h training (left bar graph). Discussion h k However, to assure that a manipulable “mental image” of the soundscape-face is really constructed, future studies should specifically investigate whether properties of face-shape perception known in the sighted population, extend to the blind population perceiving face- shapes via SSD (e.g., changing in view-points), and whether to perceive auditory face-shapes participants rely more on object-based mechanisms or utilize approaches more typical of visual face perception, such as holistic face processing21. Recently it has been shown that in some instances, interaction with human voices or tactile https://doi.org/10.1038/s41598-022-08187-z Scientific Reports \| (2022) 12:4330 \| www.nature.com/scientificreports/ https://doi.org/10.1038/s41598-022-08187-z Scientific Reports \| (2022) 12:4330 \| www.nature.com/scientificreports/ addition of color and specifically observed to facilitate face recognition15. Even though these visual results can- not be directly projected to the current (auditory) condition, we propose that our auditory results suggest that color transformation aided our participants in the correct perception of all face parts, and ultimately of whole face-shapes. Specifically, we suggest that the addition of the ‘color’ feature to soundscapes might have provided another discrimination feature among auditory pixels, and thus might have increased discriminability among face features. Crucially, these results are in line with another recent work from our team which similarly showed that color-to-timbre mapping enhanced discrimination of auditory letters and boosts reading performance via the EyeMusic SSD compared to identical monochrome soundscapes16. y p p Future studies exploring the advantages of auditory SSD-conveyed colors by directly comparing colorful to monochromatic complicated soundscapes will advance our understanding of the extent to which the visual domain compares to the auditory domain in image perception. Face‑shapes identification in the inverted (untrained) orientation. The successful identification of face-shapes in their untrained, inverted orientation suggests participants were able to build a mental representa- tion of the visual information embedded within a soundscape that they could manipulate (e.g., rotate) to match its upright and trained orientation. The process by which participants were able to generalize face identification to the inverted orientation could arise from two non-mutually-exclusive mechanisms: first, the extraction of features within the inverted face (blue eyes, long, blond hair etc.) and the matching of the features to the suitable upright face via feature-based matching; second, the mental rotation of the whole face-shape with its different spectral properties and the consequent matching to the corresponding face in the upright orientation. Mental rotation of objects is a skill that develops through life, with indications of its facilitation via visuo-haptic object manipulation34,35. Mental rotation of objects has been linked to motor experience, hormones, and experience- dependent processes36–38. Previous studies showed that blind individuals, even those who acquired blindness at birth and thus have no visual experience, are able to mentally rotate objects perceived through touch and are not deficient in their mental rotation abilities compared to sighted adults performing the same task also through touch39–41. Overall, the current results strengthen previous findings concluding that both the construction of a mental image and its rotation are not limited to information acquired via the visual modality. www.nature.com/scientificreports/ Yet unlike the tactile modality which is commonly used for tactile recognition of objects by both sighted and blind, the audi- tory sensory modality does not commonly provide information about objects’ shapes or orientation. Our study thus expands previous findings by providing initial results suggesting that the building of a mental image and its complex spatial manipulation can also be achieved via the non-traditional auditory modality.i y y Interestingly, studies on face identification in sighted individuals show, similarly to the present results, that the identification of a face in its inverted form results in a drop in both accuracy and identification speed termed the “face inverted effect”42. Such an effect has been shown to be especially enhanced when configural aspects of the faces were manipulated (i.e., when faces differed among themselves in terms of spatial features such as the spacing between nose and mouth) compared to featural or contour manipulations6. Future studies should investigate whether SSD face identification in the blind is based on the same mechanism by also employing tra- ditional paradigms typically used in the sighted population to investigate face inversion, such as the composite effect21. These works will advance our understanding of the processes underlying SSD face-shapes learning and generalization. In addition, and more generally, such studies will advance our understanding of the properties and constraints characterizing orientation-manipulations via the auditory modality. Conclusions In this work, we showed for the first time that congenitally blind adults can successfully perform whole-face- shapes identification via the auditory modality using a shape and color preserving visual-to-auditory SSD. We show that in the context of a dedicated training program, congenitally blind adults are able to perceive compli- cated soundscapes, and despite the auditory complexity of the sound, they are able to perceive detailed features embedded within the image and use this information to correctly identify face-shapes. Moreover, although shape identification and its orientation in space are not commonly performed using the auditory modality in daily life, and despite the completely different acoustic properties of the untrained, inverted face-shapes compared to the trained, upright faces, we found that congenitally blind adults are able to generalize face-shape identity to the inverted (untrained) orientation. Finally, we show that once the initial intense 12-h face training was concluded, our SSD-expert participants were able to extend the concepts learned during training to a new cohort of faces; they could successfully learn a set of new face-shapes within 2 h of additional tailored training. On the behavioral front, our results show the feasibility of the use of SSD to convey complicated visual objects. On the clinical front, our results encourage the use of SSD as a rehabilitative tool both for the blind and for the visually restored populations. On the neural front, our results produce interesting hypotheses for face processing, auditory processing, and cross-modal plasticity in the congenital absence of visual input. www.nature.com/scientificreports/ This result may further encourage the introduction of SSD-based training as a rehabilitation tool for the blind as well as aid in sight restoration rehabilitative programs, by suggesting that with increased exposure, SSD-related learning time can become shorter. Future studies should further investigate the mechanisms mediating this enhanced perfor- mance, including the generalization of trained principles to novel stimuli as well as the role of memory during the learning process. Indeed, we cannot exclude that our blind participants may have learned the main distinct features of the shortly-trained face-shapes soundscapes, and may have used them to memorize the soundscapes. Perception of multi‑color, complicated images via the auditory modality. The translation of colorful cartoon faces via the EyeMusic SSD into the auditory modality results in complicated soundscapes. Because of the sweep-line technique used by EyeMusic and other visual-to-auditory SSDs12, up to 30 musical tones of different timbres representing up to 30 visual pixels are played in sync. However, our results suggest that a training program aimed to specifically teach the perception of multiple pixels at the same time, namely one of the biggest challenges of visual-to-auditory SSD perception19,32, can result in good integrational ability. This was demonstrated by participants’ successful recognition of complicated face-shapes soundscapes, as well as by their detection of details embedded within the soundscapes that we assessed during training. This result suggests that to improve the usability of SSDs for the blind, training programs with specific objectives and dedicated training approaches should be designed to achieve specific rehabilitation outcomes. pp g pi One additional important aspect of these results is that they suggest that color, in the form of color-to- timbre mapping, may have exerted a facilitatory role on the learning of these complex soundscapes. Indeed in vision, color has been found to enhance functional acuity or in other words, helped in solving complex visual tasks33. Indeed in the context of visual tasks, the identification of low-resolution objects is enhanced by the Scientific Reports \| (2022) 12:4330 \| https://doi.org/10.1038/s41598-022-08187-z www.nature.com/scientificreports/ Methods Participants. Notably, and in accordance with the informed consent process, participants could decide to participate in only a subset of the experimental proce- dure without providing us with a reason. Please note that since not all participants took part in all aspects of the training program, we report the number of participants that performed each part of the experimental protocol. Tools. EyeMusic algorithm. The EyeMusic visual-to-auditory SSD was used to teach participants to identify whole-face shapes, as well as words and hand gestures (see details in the following section). EyeMusic transforms each pixel of a given image into what we term auditory soundscapes, an auditory pattern preserving shape, color, and spatial layout of objects. In brief, the EyeMusic algorithm down-samples each image to 50*30 pixels. Then, a sweep-line approach transforms each pixel in a given image into a corresponding sound. First, the x-axis is mapped to time; each image is scanned column-by-column from left to right, such that pixels on the left side of the image are played before those on the right. Second, the y-axis is mapped to pitch variations using the penta- tonic scale, such that the lower the pixel in the image, the lower the corresponding pitch sonifying it. Third, color is conveyed through timbre manipulations, such that each color is played using a different musical instrument. EyeMusic has five colors (white, green, red, blue, and yellow), and black is mapped as silence (Fig. 1). Brightness levels are conveyed via sound volume variations (for full details, see Abboud et al.11). During both the training and experiments, Eye-Music soundscapes lasted 2.5 s. EyeMusic training sessions. Face identification via shape recognition is a visually-dominant skill with which congenitally blind adults have little if any experience. Before training, our congenitally blind participants were largely unaware of face-related shapes; thus, a crucial aspect of our structured training program was famil- iarization with this novel object category. Furthermore, auditory face identification is an extremely challenging behavioral task because of the complexity of the soundscapes created by faces. Therefore, our training program focused on how to interpret such complex auditory soundscapes, specifically the perception of multiple tones played at sync.i Participants in all experiments were already proficient EyeMusic users who had extensive previous training with the device (> 50 h) before face training began. Methods Participants. Participants. 12 congenitally blind participants (four men, average age 36 years) with no reported neuro- logical conditions participated in the experiments. All participants had extensive experience (50–70 h of train- ing) with EyeMusic or other visual-to-auditory SSDs before starting the experiment. For detailed characteristics of the participants, see Table 1.h The Hadassah Medical Centre Ethics Committee approved the experimental procedure; written informed consent was obtained from each participant. All methods were performed in accordance with the relevant guidelines and regulations. Participants were reimbursed for their participation in the study. The participant who was involved in the movie demonstrating auditory face identification provided their informed consent for the https://doi.org/10.1038/s41598-022-08187-z Scientific Reports \| (2022) 12:4330 \| www.nature.com/scientificreports/ Table 1. Demographic details of participants. Participant Age Blindness cause Light perception Age at blindness onset Braille reading Handedness Participation in Part I: Interim testing Part II: face identification Part III: rapid face training FO 33 Microphthalmia No 0 Yes (since age 5) Right V V ElMa 36 Retinopathy of prematurity No 0 Yes (since age 5) Right V JH 42 Leber’s disease Faint 0 Yes (since age 5) Ambidextrous V V V NN 45 Retinopathy of prematurity No 0 Yes (since age 6) Right V V V PC 41 Retinopathy of prematurity No 0 Yes (since age 6) Right V V V PH 42 Rubella No 0 Yes (since age 5) Right V V FN 33 Leber’s Disease Faint 0 Yes (since age 5) Right V V DS 34 Retinopathy of prematurity No 0 Yes (since age 6) Right V V UM 39 Retinoblastoma No 3 Yes (since age 4) Ambidextrous V V HB 27 Anophtalmia, fall No < 1 Yes (since age 4) Left V V V UM 33 Anophtalmia No 0 Yes – V DK 27 unknown No No Yes (since age 4) Ambidextrous V Table 1. Demographic details of participants. Table 1. Demographic details of participants. publication of identifying information on an online open-access platform. Notably, and in accordance with the informed consent process, participants could decide to participate in only a subset of the experimental proce- dure without providing us with a reason. Please note that since not all participants took part in all aspects of the training program, we report the number of participants that performed each part of the experimental protocol. publication of identifying information on an online open-access platform. www.nature.com/scientificreports/ to identify each whole face and notice details embedded within the face when it was played as a whole. At the end of training, a subset of the cohort (nine participants, five of whom also took part in the interim test at the end of Part 1) took part in four experiments testing face identification, face-feature detection, discrimination of faces between the upright (trained) or inverted orientation (untrained), and identification of faces in the inverted orientation (untrained). to identify each whole face and notice details embedded within the face when it was played as a whole. At the end of training, a subset of the cohort (nine participants, five of whom also took part in the interim test at the end of Part 1) took part in four experiments testing face identification, face-feature detection, discrimination of faces between the upright (trained) or inverted orientation (untrained), and identification of faces in the inverted orientation (untrained). Part 3: Rapid learning of six new faces. To test whether principles learned during the face-training program could be easily generalized to the study of additional face exemplars, a subset of nine participants from the cohort participated in an additional experiment in which they learned six new cartoon faces during 2 h of additional training. Testing was repeated before and after training on the discrimination of trained vs novel, untrained faces and on the identification of newly trained faces (Fig. 2). Training procedure and experiment. Part 1: Face‑feature perception—initial training and interim (explorative) test. Part 1a: Face training. To address the soundscapes’ complexity, the first part of the train- ing program consisted of three sessions of two hours each, specifically designed to teach participants the com- mon details of faces and the perception of complex soundscapes. First, participants were introduced to six car- toon faces adapted from the children’s game “Guess Who” and translated into soundscapes using EyeMusic (see Fig. 1). In the first stages of the training, and for each face separately, participants learned to interpret separate horizontal strips of the images (bottom, top, and middle), to gradually advance their skill so they could focus on perceiving small details embedded within the complex sounds. p g p In more detail, each visual image of a cartoon face was cropped into three strips—top, middle, and bottom portion of the image (Fig. 2). www.nature.com/scientificreports/ Participants were instructed to listen carefully to the first strip, the top strip, and to detail as much as possible the colors and shapes embedded within the strip. Once details were given, the participant and instructor discussed the details’ possible implication for the face, such as: “Could the color you heard represent hair? Or is it skin-color that represents a bald head? Can hair be heard all along the strip, or only at the beginning and the end, representing a bald patch?” Then, the middle portion of the face was played; once again, the participant was requested to detail as much as possible the different tones within the strip. With the help of the instructor, the participant was instructed to concentrate on specific parts of the image, such as notes representing eyes, and in some cases, the absence of notes. The bottom strip was played in the same way. After participants learned all three strips, the complete soundscape was introduced, and participants were instructed to integrate the details from the strips into the perception of the complete soundscape. With the help of the instructor, and by comparing the complete soundscape to the strips, participants were able to “hear” the features they learned to identify in the strips, while listening to the complete soundscape. During the presentation of face-shapes we introduced and discussed in more depths some concepts related to face-shapes participants had only brief and descriptive knowledge of. For instance, the concept of eyebrows and their location as framing the eyes, the components of the eyes such as the pupils, the fact that irises are a round shape within the larger eye and differ in color among different people (and on top of that, that the colorful part of the eye, contains another round, black shape—the pupil), that eyebrows and hair usually share color, the general difference in eyebrows thickness between men and women, the fact lips are a distinct facial feature, thanks to their contrasting color from the skin of the face, the relation between glasses and the eyes (the transparent lenses with a colorful frame). These concepts were in general unfamiliar or did not play a role in participants’ lives before the experiment. www.nature.com/scientificreports/ These discussions raised some interesting questions from our participants, and also connected them to some social norms and conventions (such as the perception that a light eye-color is considered unique and beautiful in Israel; the idea of women trimming their eyebrows; the use of makeup to elongate eyelashes and make lips “pop” against the skin; coloring ones’ hair etc.). In this part of training, participants, with the help of the instructor, also concentrated on the spatial rela- tionship between face parts. Although they had the semantic knowledge on the position of the eyes “above” the nose, and the mouth “below” the nose, the use of three horizontal strips, rather than separate isolated features, facilitated and strengthened the understanding of this spatial configuration. Indeed, we qualitatively observed that with each additional learned face, participants grew more comfortable in their skills and could more quickly describe the exemplar.t p After participants were comfortable with their ability to perceive features and could describe in detail the face they were hearing, the instructor provided a name for the just-learned face. This was done to limit the possibility that the participant would simply match an abstract sound to a name rather than perceive the soundscape with its details. Notably, although during this phase we presented portions of faces, we never trained single facial features in isolation: each strip contained multiple facial features or parts of features (e.g., each top strip contained hair, eyes, glasses etc.). This is very different from tactile exploration where each facial feature is generally explored alone, separately from the others. We assigned a name to the cartoon because it has been shown that assigning names to new faces improves face recognition skills43. In addition, doing so gave participants with a sense of achievement; they could recognize specific faces that had been inaccessible before training. After three 2-h ses- sions, participants completed the interim test. Part 1b: Interim explorative test after six hours of face training. Assessment 1: “Guess Who” game. We per- formed an interim test to qualitatively monitor face-training progress. Using the “Guess Who” children’s game, we asked each participant yes/no questions on the facial features of each of the six faces, for example: “Does this character have blue eyes?” “Does this character wear glasses?” The purpose of this test was threefold. Methods Participants. Importantly, unlike previous training procedures which con- sisted mostly of line drawings of simple shapes, this was the first time participants were presented with images filled with color and with colorful features embedded within the images whose interpretation was crucial to succeed in the tasks. The difference between line drawing and color-filled images, when translated to EyeMusic, is a difference between the perception of 1–2 tones and the perception of 20–30 tones, with differences in timbres between tones reflecting colorful facial features. Through the training program, participants learned to extract the meaningful differences among tones played at the same time and to construct a comprehensive perception of the image through its soundscape. Experimental procedure. Part 1: Face‑feature perception—initial training and interim (explorative) test. EyeMusic experts underwent six hours of face training focused on the learning of common facial features and the perception of complicated full-face soundscapes. At the end of this first training, a subset of the cohort (seven participants) took part in an interim face-feature identification test to check and quantify their facial- feature perception to ensure the efficacy of the training program and face-perception progress. Part 2: Additional training and systematic testing. The second part of training, consisting of six additional hours, focused on the identification of six cartoon faces. This part of the training focused on participants’ ability https://doi.org/10.1038/s41598-022-08187-z Scientific Reports \| (2022) 12:4330 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Part 2: Additional training and systematic testing. Part 2a: Face training. By the end of Part 1 of training, par- ticipants were largely able to answer yes/no questions on the faces and identify changes in color of the features within the learned faces. Yet because of the complexity of the soundscapes, participants were not proficient at identifying the faces or discriminating among faces with similar visual features (e.g., long-haired women). Thus, the second part of training, consisting of three additional two-hour sessions, focused on the perception of complete soundscapes of faces using several training tasks. One task was to identify each complete face, without first listening to the strips composing it. Another was to listen to two faces played in an interwoven manner and identify both faces. This task helped participants note specific differences between similar faces. For instance, the three presented women had similar hair color; thus, to distinguish among them, participants had to focus on the sound amplitude (volume) representing how bright or dark each woman’s hair was. In this part of training, participants were encouraged to openly describe the faces. Part 2b: Testing face identification and generalization to untrained orientation (inverted) after 12 h of train- ing. All experiments in parts 2 and 3 were performed using Presentation software (Version 18.0, Neurobehav- ioral Systems, Inc., Berkeley, CA, www.neurobs.com).i Part 2b: Testing face identification and generalization to untrained orientation (inverted) after 12 h of train- ing. All experiments in parts 2 and 3 were performed using Presentation software (Version 18.0, Neurobehav- ioral Systems, Inc., Berkeley, CA, www.neurobs.com).i y y Experiment 1: Face identification. First, to ensure success of training, especially participants’ ability to iden- tify each of the learned six faces, we conducted a basic face identification experiment. Participants were asked to name each of the six experimental faces they received training on. Each character was played repeatedly until participants named it (roughly within two repetitions). Each character was presented in 16 separate trials, 96 trials overall, presented in random order. Participants provided their responses verbally, and the experimenter inserted them in the computer. The rate of correct responses and reaction times were analyzed. ph p y Experiment 2: Upright vs. inverted (untrained) orientation discrimination. www.nature.com/scientificreports/ Participants were allowed to listen to the original soundscape and the manipulated soundscape to compare them before giving their answer. This task was very intense and challenging for participants, as it required a lot of focus to answer correctly, given the complexity of the soundscapes and the small manipulations we introduced. Stimuli were presented using the EyeMusic SSD. Each stimulus was played, either continuously or in an interwoven fashion with the original soundscape, until participants provided a response. Specifically, participants were asked to respond on whether they detect a change between the two soundscapes And if yes, to localize the This experiment included only 12 trials. We limited the number of trials because only a limited number of facial features and colors could be manipulated in a meaningful way. We only included color-related manipula- tions. Participants were allowed to listen to the original soundscape and the manipulated soundscape to compare them before giving their answer. This task was very intense and challenging for participants, as it required a lot of focus to answer correctly, given the complexity of the soundscapes and the small manipulations we introduced. Stimuli were presented using the EyeMusic SSD. Each stimulus was played, either continuously or in an interwoven fashion with the original soundscape, until participants provided a response. Specifically, participants were asked to respond on whether they detect a change between the two soundscapes. And if yes, to localize the change (i.e., in which face feature) and identify the changed color. Responses were registered by the experimenter. We assessed the accuracy of detected changes, the accuracy of localizing the changes (how accurately participants identified the specific feature that was changed, or the location of the change within the spatial soundscape), and the accuracy of identifying new colors. Stimuli were presented using the EyeMusic SSD. Each stimulus was played, either continuously or in an interwoven fashion with the original soundscape, until participants provided a response. Specifically, participants were asked to respond on whether they detect a change between the two soundscapes. And if yes, to localize the change (i.e., in which face feature) and identify the changed color. Responses were registered by the experimenter. We assessed the accuracy of detected changes, the accuracy of localizing the changes (how accurately participants identified the specific feature that was changed, or the location of the change within the spatial soundscape), and the accuracy of identifying new colors. www.nature.com/scientificreports/ First, we wanted to confirm the training was effective, and participants had learned to identify facial features embedded in the soundscapes. Second, we wanted to ensure the participants had learned and focused on all facial features, https://doi.org/10.1038/s41598-022-08187-z Scientific Reports \| (2022) 12:4330 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ thus preventing face recognition based on a simple sound-to-name matching, Third, we wanted to give par- ticipants an opportunity to apply their acquired skills to a more ecological and fun activity they were unable to participate in prior to training—the blind are generally excluded from this popular children’s game.h thus preventing face recognition based on a simple sound-to-name matching, Third, we wanted to give par- ticipants an opportunity to apply their acquired skills to a more ecological and fun activity they were unable to participate in prior to training—the blind are generally excluded from this popular children’s game.h This experiment included 32 trials, containing yes/no questions about the faces, plus six naming trials (one naming trial per cartoon face). The questions addressed both large visual facial features (16 questions about hair, etc.) and smaller facial features (16 questions about eyes, glasses). Stimuli were presented using the EyeMusic SSD, and each stimulus was played until an oral response was given by the participant. Responses were regis- tered by the experimenter. Rate of correct responses for yes/no answers was analyzed. As a secondary result, the percentage of correctly identified characters was also assessed. p g yi Assessment 2: Change detection. To further test participants’ ability to recognize features embedded within the complicated soundscape, we performed a “change detection” task in which the color of one feature of the image was changed (e.g., the eye-color; hair color, etc.). Our aim was to see how well facial features embedded within the soundscapes were detected by participants by testing whether they were able to detect changes made to these features.h This experiment included only 12 trials. We limited the number of trials because only a limited number of facial features and colors could be manipulated in a meaningful way. We only included color-related manipula- tions. Participants were allowed to listen to the original soundscape and the manipulated soundscape to compare them before giving their answer. This task was very intense and challenging for participants, as it required a lot of focus to answer correctly, given the complexity of the soundscapes and the small manipulations we introduced. This experiment included only 12 trials. We limited the number of trials because only a limited number of facial features and colors could be manipulated in a meaningful way. We only included color-related manipula- tions. www.nature.com/scientificreports/ We thus taught participants to recognize six unfamiliar faces, in two hours of training. Part 3b: Assessment of learning. To assess learning, we performed the following experiments.h Experiment 4 (pre-post training). This experiment involved the discrimination of previously trained faces vs. unfamiliar faces (untrained). Before training, we tested participants on a discrimination task of all 12 faces; six were already familiar from previous training, and six were new. Participants were asked to classify if a face belonged to the cohort of familiar faces (i.e., trained faces) or not (i.e., untrained), and provide their response by pressing one of two possible response keys with the index and ring fingers. Each face was repeated continuously until a response was provided. Each face was repeated six times for a total of 72 trials presented in random order. The number of correctly classified faces and reaction times were analyzed. This task was performed to ensure that despite deliberate visual similarities among the familiar and unfamiliar faces, the resulting soundscapes were different enough to be distinguished by our participants. f g g y p p We repeated the experiment to assess improvement in discrimination following training on the six new faces. The experiment was the same, but this time, participants were instructed to discriminate each face as belonging to either the “old cohort” or the “new cohort” learned that day.i y Experiment 5. We conducted a face identification task with the six newly-trained faces. Participants named each of the six characters learned during the final 2-h face-training session. Each character was played repeat- edly until participants named it (roughly within two repetitions). Each character was presented in 12 separate trials, 96 trials overall, in random order. Each face soundscape was played continuously until a response was provided. Participants were instructed to press the space bar when they knew the answer and then to provide their responses verbally. The experimenter entered responses into the computer. The rate of correct responses and reaction times were analyzed. Data analysis. Data were analyzed using JASP (version number 0.14). We used a paired t-test when com- paring results for different conditions (upright/inverted or pre-post,) and a t-test against chance level when comparing conditions to chance. Chance level was set at random, since participants had no previous experience with face soundscapes, and were unable to interpret the soundscapes, or even extract meaningful information regarding the larger features prior to training. www.nature.com/scientificreports/ using EyeMusic in the untrained, inverted (upside down) orientation. New faces included six visually similar faces not introduced during training. Scrambled faces included the six familiar faces in a scrambled form. The faces were divided into nine parts and scrambled randomly using MATLAB. The resulting images were sonified via EyeMusic. The scrambled faces condition was included as a motor control, and we do not discuss results for this condition in this paper.h The conditions were presented in separate blocks. Each condition was repeated six times, in a pseudorandom order, for a total of 24 blocks. The experiment was repeated for four runs, for a total of 96 blocks. Each exemplar was repeated eight times. In each block, two different stimuli belonging to the same experimental condition were displayed, each lasting five seconds (two consecutive repetitions of 2.5 s per stimulus), followed by a response interval of 2 s. Each block started with an auditory cue indicating the tested category and lasting two seconds (trained faces, inverted, new, scrambled). Participants provided their responses using a response box at the end of both repetitions of a stimulus. For trained (upright) and inverted conditions, participants were instructed to identify the character. To limit the number of response keys thus allow one-handed response, in two of the four runs, only female characters were presented, while in the other two runs, only male characters were presented. As a result, chance level in this section is 30%. In total, each block lasted 16 s and was followed by a 10 s rest interval. For new faces, participants performed a feature detection task; they were instructed to listen to each soundscape carefully. If they could identify yellow features, they were instructed to press one button, and if not, to press another button. Digital auditory soundscapes were generated on a PC, played on a stereo system, and transferred binaurally to participants through a pneumatic device and silicone tubes into commercially avail- able noise-shielding headphones. For all experiments, accuracy of responses was analyzed. In this work, we only present results for the trained and inverted conditions. The rest of the results from the scanning session will be presented elsewhere. Part 3: Rapid learning of six unfamiliar faces. Part 3a: Learning of new faces. Following the in-depth learning of the six faces, we wanted to test whether learning of new face examples would become faster. www.nature.com/scientificreports/ Second, to ensure training had taught participants to understand face shapes and composition and to test whether participants were able to recognize face orientations that were not trained (i.e., inverted), we conducted an orientation-discrimination task. During the discrimination task, a character was repeatedly played in either the standard upright (trained) or inverted (up-side down) orientation (untrained) until participants assigned an orientation to the soundscape. Participants used two response keys to register their answers (either upright or inverted). Each character in each orientation was presented six times (six separate trials) for a total of 72 trials presented in random order. The rate of correct responses and reaction times were analyzed.ih p y Experiment 3: Identification of faces in upright and inverted orientations. This experiment was conducted while participants were lying in an MRI machine. We wanted participants to be naive to the identification task of inverted faces and perform it for the first time while being scanned, Therefore, we conducted this experiment in the scanner. Note that our neuroimaging results will be reported in a separate paper. In vision, the inversion of a face does not change the visual attributes of the face itself, only their position in space, but in the auditory modality, the inversion of a face completely changes the acoustic properties of the soundscape. The ability to identify characters in their inverted orientation would show that participants were able to recognize characters based on their unique characteristics, not by simply matching sound to an arbitrary name, and could also perform mental rotation of the soundscapes’ representations. p p We used four sets of face stimuli: trained faces, trained faces in the untrained, inverted orientation (inverted faces), entirely new, untrained faces (new faces), and scrambled faces. Trained faces included the six colorful faces participants learned to identify during training. Inverted faces included each of the six trained faces sonified Scientific Reports \| (2022) 12:4330 \| https://doi.org/10.1038/s41598-022-08187-z www.nature.com/scientificreports/ Received: 10 August 2021; Accepted: 22 February 2022 References 1. Simion, F., Di Giorgio, E., Leo, I. & Bardi, L. The processing of social stimuli in early infancy. From faces to biological motion perception. 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Vis. cogn. 15, 564–577 (2007).hf p g g 8. Teunisse, J. P. & De Gelder, B. Face processing in adolescents with autistic disorder: The inversion and composite effects. Brain Cogn. 52, 285–294 (2003). g 9. Pietrini, P. et al. Beyond sensory images: Object-based representation in the human ventral pathway. Proc. Natl. Acad. Sci. USA 101, 5658–5663 (2004). ( ) 10. Kim, J.-K. & Zatorre, R. J. Generalized learning of visual-to-auditory substitution in sighted individuals. Brain Res. 1242, 263–275 (2008). 1. Abboud, S., Hanassy, S., Levy-Tzedek, S., Maidenbaum, S. & Amedi, A. EyeMusic: Introducing a “visual” colorful experience fo the blind using auditory sensory substitution. Restor. Neurol. Neurosci. 32, 247–257 (2014). Author contributions R.A. and A.M. conceived the experiments. R.A. acquired the data. R.A. and B.H. analyzed the data. R.A., B.H. and A.M. wrote the paper. All authors reviewed the manuscript. https://doi.org/10.1038/s41598-022-08187-z Scientific Reports \| (2022) 12:4330 \| www.nature.com/scientificreports/ Funding This study was funded by Na’amat Scholarship, European Research Council (773121), James S. McDonnell Foundation (220020284), Joy Ventures Grant and European Union GuestXR. Competing interests The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-022-08187-z. Correspondence and requests for materials should be addressed to R.A. 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. 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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 Author(s) 2022 https://doi.org/10.1038/s41598-022-08187-z Scientific Reports \| (2022) 12:4330 \|
https://openalex.org/W2777155899	https://europepmc.org/articles/pmc5796356?pdf=render	English	null	Mixed H2/H∞-Based Fusion Estimation for Energy-Limited Multi-Sensors in Wearable Body Networks	Sensors	2,017	cc-by	12,865	Chao Li 1,2, Zhenjiang Zhang 3,* and Han-Chieh Chao 4,5,6,7 Chao Li 1,2, Zhenjiang Zhang 3,* and Han-Chieh Chao 4,5,6,7 1 Key Laboratory of Communication and Information Systems, Beijing Jiaotong University, Beijing 100044, China; 15111037@bjtu.edu.cn 2 Beijing Municipal Commission of Education, Department of Electronic and Information Engineeri Beijing Jiaotong University, Beijing 100044, China j g g y j g 3 School of Software Engineering, Beijing Jiaotong University, Beijing 100044, China 3 School of Software Engineering, Beijing Jiaotong University, Beijing 100044, China 4 School of Information Science and Engineering, Fujian University of Technology, Fuzhou 350118, Chin hcc@mail.ndhu.edu.tw 5 School of Mathematics and Computer Science, Wuhan Polytechnic University, Wuhan 430024, China 7 Department of Computer Science and Information Engineering, National Ilan University, Yilan 26047, Taiwan * Correspondence: zhangzhenjiang@bjtu.edu.cn; Tel.: +86-133-0111-7395 Received: 21 September 2017; Accepted: 24 December 2017; Published: 27 December 2017 ceived: 21 September 2017; Accepted: 24 December 2017; Published: 27 December 2017 Abstract: In wireless sensor networks, sensor nodes collect plenty of data for each time period. If all of data are transmitted to a Fusion Center (FC), the power of sensor node would run out rapidly. On the other hand, the data also needs a ﬁlter to remove the noise. Therefore, an efﬁcient fusion estimation model, which can save the energy of the sensor nodes while maintaining higher accuracy, is needed. This paper proposes a novel mixed H2/H∞-based energy-efﬁcient fusion estimation model (MHEEFE) for energy-limited Wearable Body Networks. In the proposed model, the communication cost is ﬁrstly reduced efﬁciently while keeping the estimation accuracy. Then, the parameters in quantization method are discussed, and we conﬁrm them by an optimization method with some prior knowledge. Besides, some calculation methods of important parameters are researched which make the ﬁnal estimates more stable. Finally, an iteration-based weight calculation algorithm is presented, which can improve the fault tolerance of the ﬁnal estimate. In the simulation, the impacts of some pivotal parameters are discussed. Meanwhile, compared with the other related models, the MHEEFE shows a better performance in accuracy, energy-efﬁciency and fault tolerance. Keywords: fusion estimation; wearable sensors; energy-efﬁciency; accuracy sensors sensors sensors 1. Introduction Recent technology advances have led to the development of sensing, computing, and communication equipment, which are becoming an essential part of our daily lives. These ubiquitous systems have proved to be effective in numbers of domains from medical and well-being to the military and smart vehicles. Wearable body networks (WBNs) [1,2] are one of these platforms consisting of enormous numbers of body sensor nodes. These nodes are a type of portable device that can be worn directly or integrated into the user’s clothes. Meanwhile, using different uploaded applications, WBNs can realize many useful functions, such as monitoring blood glucose, blood pressure, and heart functions in the medical ﬁeld; motion monitoring in nursing and sports; and data collection in industry and the military [3,4]. Sensors 2018, 18, 56; doi:10.3390/s18010056 www.mdpi.com/journal/sensors www.mdpi.com/journal/sensors Sensors 2018, 18, 56 2 of 19 Nowadays, wearable devices use microminiaturization and multi-functionalization, which can collect a great deal of data while maintaining their small size. Therefore, multi-sensor fusion estimation [5,6], which has attracted considerable research interest during the past decades, is recommended for managing the resulting multiple data streams. Meanwhile, several kinds of challenges need to be overcome in this ﬁeld, such as power efﬁciency, memory storage, and connectivity. As a general rule, WBNs are battery-operated. The wearable devices need to collect the body data and transmit them to the Fusion Center (FC). However, the more data are transmitted, the longer the time needed to process them is. On the other hand, the main energy consumption in the battery-operated wearable device is data transmission. Meanwhile, most communication networks can only carry a ﬁnite amount of information per period. Thus, energy efﬁciency is considered one of the most important challenges [7] to address the limitation of energy consumption. Here, a normal idea is to reduce communication trafﬁc between sensors and the FC at each period. Therefore, two common methods have been used in existing research: the dimensionality reduction method [8,9] and the quantization method [10]. The idea of the dimensionality reduction method is used to convert high-dimensional data into low-dimensional data through speciﬁc mechanisms. Meanwhile, in the quantization method, the number of coding bits for each component of high-dimensional data is reduced, and the dimension of the quantized data is the same as that of the original data. Furthermore, in most cases, the collected data contain noise that comes from the wearable devices themselves and environmental effects. 1. Introduction Therefore, a state ﬁlter is employed to output the estimation data with more accurate and reliable. One of the typical ﬁlters is the Kalman Filter (KF) [11] which can estimate the states of linear Gaussian state-space models. However, the KF-based [12,13] ﬁlters usually rely on the assumption that the noise should follow the Gauss process or Gaussian sequence, which is hard to satisfy in most practical applications. Actually, the KF-based ﬁlters need the expectation and variance of the noise to estimate the status values. Therefore, when the noise has bounded energy and the statistical property of the noise is unknown, the KF-based ﬁlters couldn’t be used for estimation. The H∞estimation theory [14] was introduced to solve this state estimation problem. In the H∞estimation theory, the status value is obtained according to the maximum amplitude of the evaluated error. On the other hand, H∞fusion estimation may be conservative and lead a large intolerable estimation error variance when the system is driven by white noise signals. Then, the mixed H2/H∞estimation theory [15,16] is used in this technical area, which can reduce the estimation error with the bounded noise. This paper proposes a novel energy-efﬁcient fusion estimation model, which uses the mixed H2/H∞ﬁlter to remove the noise, and combine the dimensionality reduction method and the quantization method to compress the collected data. The proposed model can regulate the number of transmitted data according to the thresholds, and prolong the lifetime of WBNs. The remainder of this brief is organized as follows: Section 2 introduces related works in the fusion estimation ﬁeld. Section 3 describes the preliminary work. Then, a novel mixed H2/H∞-based energy-efﬁcient fusion estimation (MHEEFE) model is proposed and described in Section 4. Next, some important parameters are analyzed in Section 5. Experimental results, including comparisons, are presented in Section 6. Finally, the conclusion is given in Section 7. 2. Related Works H2/H∞-based ﬁlter comes from the H control theory, which obtains the controller according to the inﬁnite paradigm-based optimization of some performance indicators in Hardy space. In these ﬁlters, H2/H∞-based ﬁlter is an important method in the state estimation, which offers much better robustness in performance when the noise has bounded energy and the statistical property of noise is unknown. In other words, the H2/H∞-based ﬁlter does not need the speciﬁc statistical properties of noise, but describes the noise with bounded energy. In the preliminary research, a number of H2/H∞-based controllers or ﬁlters were proposed in different systems, such as the integral quadratic constraints system [17], stochastic uncertain 3 of 19 Sensors 2018, 18, 56 systems [18], uncertain stochastic time-delay systems [19], aerospace system [20] and polytopic discrete-time systems [21]. Afterwards, kinds of the H2/H∞-based fusion ﬁlters were presented, which are not only used to estimate the collected data, but also combined the data fusion method [22,23], to increase the accuracy of the ﬁnal estimate. In [23], Wen et al. addressed a H2/H∞-based fusion ﬁltering problem for networked dynamical systems, where measurements may arrive at fusion center in four different scenes and the fusion center could receive none, one, or multiple measurements in a fusion period. They proposed a uniﬁed ﬁnite horizon H2/H∞-based ﬁltering method to solve this problem. On the other hand, the data fusion methods are widely applied to the Internet of Things. Therefore, dozens of related studies came out of WBNs naturally. In WBNs, one of most important constraints is the limited energy, which means that the energy-efﬁciency must be considered. In WBNs, the energy is mainly the cost of data communication, and many algorithms were presented to decrease the communication trafﬁc. Zhang et al. [24] proposed an energy-efﬁcient transmission strategy by reducing the transmission rate of measurements from the sensor nodes to the fusion center. In the proposed strategy, all sensor nodes are divided into different groups, and only one group transmits their measurements in each transmission period. This strategy is efﬁcient to save the energy of nodes, but it’s only suitable for some speciﬁc WBNs. Then, Hao et al. [25] developed channel-aware algorithms for tracking nonstationary state processes based on reduced-dimensionality data collected by power-limited wireless sensors, which provided sensors with accurate estimates at affordable communication cost. In these two models, the amounts of transmission data are decreased. 2. Related Works Actually, the quantization method can reduce the communication consumption and satisfy the limited communication capacity. Then, in [26], Chen et al. studied the distributed H∞fusion ﬁltering problem for a class of networked multi-sensor fusion systems, which introduced a multiple ﬁnite-level logarithmic quantizer to reduce the transmission data. In conclusion, the dimensionality reduction method converts a multidimensional signal directly into a low-dimensional signal, while the quantization method reduces the number of coding bits for each component of a multidimensional signal. Obviously, the dimension of the quantized signal is the same as that of the original signal and the dimensionality reduction method may be more efﬁcient in trafﬁc reduction as compared with the quantization method. However, the quantization effect must not be ignored in a communication network. Therefore, Chen et al. [16] studied the problem of distributed fusion estimation, combined both two energy-efﬁcient methods and proposed a distributed mixed H2/H∞fusion ﬁlter (DMHFE) with limited communication capacity. However in [16], the presented model calculated the weights according to the H2/H∞ﬁlter, which are ﬁxed values, and this would reduce the accuracy and fault-tolerance of the estimation. Therefore, in this paper, a novel mixed H2/H∞based energy-efﬁcient fusion estimation (MHEEFE) model is proposed, which uses the mixed H2/H∞ﬁlter in local node to remove the noise, and combines the new dimensionality reduction method and the quantization method to decrease the communication consumption. Besides, an iteration-based weights calculation method is used to fuse the received data in FC, which provides a higher fault tolerance. Moreover in MHEEFE model, the parameters of mixed H2/H∞ﬁlter are calculated in FC, and the energy-efﬁcient strategies can reduce more communication consumption while keep a higher accuracy. 3. Preliminary Work In practical application, there are some uncertain variables, whose statistics characteristics are hardly increasing but which affect the state of the system signiﬁcantly. In contrast with the well-known Kalman ﬁlter, the Hardy space-based ﬁlter does not make any assumptions about the statistics of the 018, 18, 56 4 of 19 4 of 19 Sensors 2018, 18, 56 process and measurement noise, but only assumes that the external disturbance has bounded energy. Consider a linear discrete-time stochastic system described by the following state-space model: process and measurement noise, but only assumes that the external disturbance has bounded energy. Consider a linear discrete-time stochastic system described by the following state-space model:      x(t + 1) = Ax(t) + Bω(t) y(t) = Cx(t) + Dυ(t) z(t) = Lx(t) (1) (1) where x(t) ∈Rn is the system state, y(t) ∈Rq is the measured output, z(t) ∈Rq is the signal to be estimated, ω(t) and ν(t) are both the energy bounded signal. A, B, C, D and L are constant matrices with appropriate dimensions. Before estimation, Equation (1) should be transformed as follows: Before estimation, Equation (1) should be transformed as follows:      x(t + 1) = Ax(t) + Bf V(t) y(t) = Cx(t) + Df V(t) z(t) = Lx(t) (2) (2) where: where: V(t) = [ω(t), υ(t)]T, Bf V(t) = Bω(t), Df V(t) = Dυ(t) The key idea of the estimation problem is to ﬁnd an estimated value ˆx(t) of the signal x(t) which is satisﬁed a performance criterion preset. In mixed H2/H∞ﬁlter, that is minimized in an estimation error sense for both H2 and H∞form. The ﬁlter is based on the set of the measurement output signal obtained at each time t. In this case, the purpose is to design an asymptotically stable linear ﬁlter described by: ( ˆx(t + 1) = A ˆx(t) + K(y(t) −C ˆx(t)) ˆz(t) = L ˆx(t) (3) (3) where K ∈Rn×q is the gain matrix to be determined. 3. Preliminary Work Then, deﬁning state error as: e(t) = x(t) −ˆx(t) (4) (4) and the estimation error dynamics is given by: and the estimation error dynamics is given by: ( e(t + 1) = Ae(t) + BV(t) ez(t) = Le(t) (5) (5) where ˆA f = A −KC, ˆBf = Bf −KDf and ez(t) = z(t) −ˆz(t) The closed-loop transfer function from the noise signal V(t) to the output ez(t) is given by: The closed-loop transfer function from the noise signal V(t) to the output ez(t) is given by: HezV(ζ) ≡L(ζI −A f )−1Bf HezV(ζ) ≡L(ζI −A f )−1Bf Therefore, for the mixed H2/H∞estimation problem, determine a stable ﬁlter such that an upper bound to the H2 performance criterion is minimized and ∥HezV ∥∞≤γ. Then, A Linear Matrix Inequality (LMI) characterization is provided by Theorem 1. Theorem 1 [15]. The optimal solution of: Theorem 1 [15]. The optimal solution of: min J,Y,WTr{J} subject to: subject to: " J B′Y −D′W′ YB −WD Y # ≥0 " J B′Y −D′W′ YB −WD Y # ≥0 5 of 19 Sensors 2018, 18, 56   Y 0 A′Y −C′W′ L′ 0 γ2I B′Y −D′W′ 0 YA −WC YB −WD Y 0 L 0 0 I  ≥0 Y > 0 with Y = Y’ ∈Rn×n, W ∈Rn×r and J = J’ ∈Rm×m is such that: Tr{J} ≥∥Hezw∥2 2, ∥Hezw∥∞≤γ nd the optimal ﬁltering gain is given by K = Y−1W. and the optimal ﬁltering gain is given by K = Y−1W. and the optimal ﬁltering gain is given by K = Y−1W. For given appropriate parameters: For given appropriate parameters: 1. Determine the local estimation gains Ki in i-th sensor node; 2. Determine the parameters of the quantization method in FC, and sends them to sensor nodes; 3. Determine the parameters of the weights in FC; 4. Calculate the reorganized state estimate (RSE) according to the parameters in step 2 and 3, and send the RSE to FC for each sensor node; 5. Calculate the local compensating state estimate (CSE) according to the received RSE; 6. Calculate the ﬁnal estimate according to the weights. The proposed strategy establishes an optimal fusion criterion in terms of LMIs, which can be easily solved according to the Matlab LMI Toolbox. Besides, a dual data compression strategy (DDCS) is presented to satisfy the limited communication capacity and reduce the communication trafﬁc. However, there are some steps still can be improved. Firstly, in the procedures above, the dimensionality reduction method is executed in a random way, and for each sensor, the exact selection probabilities are given before the sensor collects the data without changing. However, in fact, in a state estimate system such as (1), ω(t) is the energy-bounded signal with uncertain statistical characteristics, then the part of system state may have a large change, meaning the energy-bounded signal outputs a large value unexpectedly. In this case, if the sensor keeps the random way to select the transmitted components, the component with large change may be missed. Therefore, a enormous error may be generated. 4. The Mixed H2/H∞-Based Energy-Efﬁcient Fusion Estimation (MHEEFE) Model 4.1. The Computation Procedures for the DMHFE with Limited Communication Capacity In [16], a distributed mixed H2/H∞fusion estimator (DMHFE) model is presented, and the computation procedures are summarized as follows: As shown in Table 1, steps 1 through 3 are used to determine the parameters. Before collecting the observation data, each sensor computes its gain matrix. The fusion center calculates the quantized parameters and sends them to each sensor. Each sensor then collects the observation data and estimates them with a mixed H2/H∞ﬁlter. After that, each sensor selects the sending components in a random way, quantizes the sending components according to the correlation parameter, and sends the RSE to the fusion center. Finally, when the fusion center receives the RSE, it compensates them to become the complete state estimate and fuse them to a ﬁnal estimate. Table 1. The computation procedures of DMHFE. For given appropriate parameters: 30 35 40 45 50 55 60 65 70 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Iterations MSE Figure 1. The mean square errors (MSEs) of distributed mixed H2/H∞fusion estimator (DMHFE). Figure 1. The mean square errors (MSEs) of distributed mixed H2/H∞ fusion estimator (DMHFE). 30 35 40 45 50 55 60 65 70 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Iterations MSE Figure 1. The mean square errors (MSEs) of distributed mixed H2/H∞ fusion estimator (DMHFE). Figure 1. The mean square errors (MSEs) of distributed mixed H2/H∞fusion estimator (DMHFE). Figure 1. The mean square errors (MSEs) of distributed mixed H2/H∞ fusion estimator (DMHFE). Figure 1 demonstrates that the wave is hard to avoid in the random dimensionality reduction method, and the difference is the time periods for the value of MSE reducing to the acceptable level. Hence, a simple experiment is performed to discuss a better dimensionality reduction method. Using the same system model above, three kinds of conditions are considered: choose the whole components, choose the components randomly and choose the components with higher probability which have a greater change. For simplicity, the MSE line for the first condition is called “Whole Line,” the second is called “Random Line,” and the third is called “Selected Line”. Then, these lines are calculated as the mean for 100 times, and the iterations from 30th to 70th are shown in Figure 2 as follows: Figure 1 demonstrates that the wave is hard to avoid in the random dimensionality reduction method, and the difference is the time periods for the value of MSE reducing to the acceptable level. Hence, a simple experiment is performed to discuss a better dimensionality reduction method. Using the same system model above, three kinds of conditions are considered: choose the whole components, choose the components randomly and choose the components with higher probability which have a greater change. For simplicity, the MSE line for the ﬁrst condition is called “Whole Line,” the second is called “Random Line,” and the third is called “Selected Line”. Then, these lines are calculated as the mean for 100 times, and the iterations from 30th to 70th are shown in Figure 2 as follows: Figure 1 demonstrates that the wave is hard to avoid in the random dimensionality reduction method, and the difference is the time periods for the value of MSE reducing to the acceptable level. For given appropriate parameters: Considering a realistic scene, in a state estimate system (2): Considering a realistic scene, in a state estimate system (2): A =   0.8673 0 0.2022 0.0293 0.9763 −0.0301 0.0259 0 0.8032  , Bf =   0.1 0.5 −0.2 0 0 0  , C = " 1 1 0 0 1 0 # , Df = " 0.3 0.2 0.1 0.3 # , L =   1 0 0 0 1 0 0 0 1  and x(0) =   0 0 0   A =   0.8673 0 0.2022 0.0293 0.9763 −0.0301 0.0259 0 0.8032  , Bf =   0.1 0.5 −0.2 0 0 0  , C = " 1 1 0 0 1 0 # , Df = " 0.3 0.2 0.1 0.3 # , L =   1 0 0 0 1 0 0 0 1  and x(0) =   0 0 0   6 of 19 Sensors 2018, 18, 56 where V(t) is the energy-bounded signal. To simplify, we deﬁne: where V(t) is the energy-bounded signal. To simplify, we deﬁne: V(t) =    h 5 5 iT , t = 50 Random Noise, others Then, the mean square error (MSE) of DMHFE is calculated, and depicted in Figure 1, which shows that when t falls between 40 and 60 there is an obvious wave. The wave occurs for the energy-bounded signal working, which the DMHFE could not avoid if the components with large change are not submitted to FC. Then, the mean square error (MSE) of DMHFE is calculated, and depicted in Figure 1, which shows that when t falls between 40 and 60 there is an obvious wave. The wave occurs for the energy- bounded signal working, which the DMHFE could not avoid if the components with large change are not submitted to FC. Then, the mean square error (MSE) of DMHFE is calculated, and depicted in Figure 1, which shows that when t falls between 40 and 60 there is an obvious wave. The wave occurs for the energy- bounded signal working, which the DMHFE could not avoid if the components with large change are not submitted to FC Figure 1. The mean square errors (MSEs) of distributed mixed H2/H∞ fusion estimator (DMHFE). For given appropriate parameters: Hence, a simple experiment is performed to discuss a better dimensionality reduction method. Using the same system model above, three kinds of conditions are considered: choose the whole components, choose the components randomly and choose the components with higher probability which have a greater change. For simplicity, the MSE line for the first condition is called “Whole Line,” the second is called “Random Line,” and the third is called “Selected Line”. Then, these lines are calculated as the mean for 100 times, and the iterations from 30th to 70th are shown in Figure 2 as follows: Figure 2. Three MSE lines. n in Figure 2, the Random Line almost maintains the largest MSE value after the energy 30 35 40 45 50 55 60 65 70 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Iterations MSEs Slected Line Random Line Whole Line Figure 2. Three MSE lines. 30 35 40 45 50 55 60 65 70 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Iterations MSEs Slected Line Random Line Whole Line Figure 2. Three MSE lines. g o Li e al o t ai tai t Figure 2. Three MSE lines. Figure 2. Three MSE lines. Sensors 2018, 18, 56 7 of 19 As shown in Figure 2, the Random Line almost maintains the largest MSE value after the energy-bounded signal works. In other words, when the signal makes a great change, the dimensionality reduction method using the random approach shows the worst performance, the Selected Line shows a better performance than Random Line, and the Whole Line is the best. Second, in the quantization method above, the quantizer is a logarithmic quantization q(x) =      ρℏu if ρℏu 1+δ ≤x < ρℏu 1−δ 0 if x = 0 −q(−x) if x < 0 (6) (6) which divides the interval closer when the value is close to 0 and incompact when the value is further from 0. However, according to (6), ρ = 0.1 and u = 2 are set. Then it results x1 = 1.1 × 10−50 and x2 = 1.1 × 10−52 are divided into different intervals. Obviously, this division is too short to quantize the continuous variable, which leads the over-division. Third, the FC calculates the weight of each sensor before the WBNs work. And those weights are ﬁxed values. 4.2. MHEEFE Model In this part, the details of the MHEEFE model is introduced, which is used for multi-sensor fusion estimation. In this model, new dimensionality reduction method and the quantization method are proposed. Moreover, an adaptive algorithm is presented to calculate the weights which can weaken the effect of data with low accuracy and keep the accuracy of ﬁnal estimate. In this section, the energy-efﬁcient data transmission strategy and the high-accuracy data fusion strategy are introduced. Before the MHEEFE model works, a mixed H2/H∞-based local ﬁlter is performed to gain the estimated value. Here, for sensor i, the state system is similar to (1): ( x(t + 1) = Ax(t) + BV(t) yi(t) = Cix(t) + DiV(t) (7) (7) where yi(t) is the observed value of sensor i, and Ci and Di are the constant matrices with appropriate dimensions. In this paper, the mixed H2/H∞ﬁlter is used for local estimation. Therefore, according to Theorem 1, the gain matrix Ki for sensor i can be calculated. Thus, the estimated value of the state is: where yi(t) is the observed value of sensor i, and Ci and Di are the constant matrices with appropriate dimensions. In this paper, the mixed H2/H∞ﬁlter is used for local estimation. Therefore, according to Theorem 1, the gain matrix Ki for sensor i can be calculated. Thus, the estimated value of the state is: (8) ˆxi(t + 1) = A ˆxi(t) + Ki(yi(t) −Ci ˆxi(x)) (8) In the mixed H2/H∞ﬁlter, the gain matrix can be conﬁrmed only by the matrix A, B, Ci and Di. Therefore, the gain matrix can be calculated at the fusion center, and then it is transmitted to each sensor before the WBNs works. In the mixed H2/H∞ﬁlter, the gain matrix can be conﬁrmed only by the matrix A, B, Ci and Di. Therefore, the gain matrix can be calculated at the fusion center, and then it is transmitted to each sensor before the WBNs works. For given appropriate parameters: However, in each time, the sensors transmit the data after being randomly selected. And the weight is larger while the transmitted data are more important. Therefore, if the accuracy of each sensor changes frequently, the weight should change too. Besides, if a sensor out of order, then it transmits a misdata. The ﬁxed weights couldn’t recognize it and may cut down the accuracy of ﬁnal estimate. Then, to solve the weaknesses above, a novel mixed H2/H∞-based energy-efﬁcient fusion estimation (MHEEFE) model is proposed. 4.2.1. The Energy-Efﬁcient Data Transmission Strategy 4.2.1. The Energy-Efﬁcient Data Transmission Strategy 4.2.1. The Energy-Efﬁcient Data Transmission Strategy In energy-efﬁcient data transmission strategy, two methods are used to reduce the communication consumption, which are the dimensionality reduction method and the quantization method. In the dimensionality reduction method, it is assumed that only ri(t) (1 ≤ri(t) < n) components of the i-th local estimated value are allowed to be transmitted to the FC in time t. Then, the selected components Sensors 2018, 18, 56 8 of 19 are quantized to the ﬁnite-level before transmitted. Here, the quantizer is similar to (6) and deﬁned by the following nonlinear mapping: q xij(t) =        ρjℏuj + sj if τ ≤ ρjℏuj 1+δj ≤xij(t) −sj < ρjℏuj 1−δj sj if xij(t) −sj < τ −q −xij(t) + sj if xij(t) −sj ≤−τ (9) (9) where uj > 0, 0 < ρj < 1, δj = (1 −ρj)/(1 + ρj) (0 < δj < 1), ¯h = 0, ±1, ±2, . . . . sj is the median of value range for the jth component of the state vector and τ is the interceptive threshold which is used to prevent the over-division. Therefore, the reorganized state estimate (RSE), which is the transmitted data for sensor i, is expressed as follows: where uj > 0, 0 < ρj < 1, δj = (1 −ρj)/(1 + ρj) (0 < δj < 1), ¯h = 0, ±1, ±2, . . . . sj is the median of value range for the jth component of the state vector and τ is the interceptive threshold which is used to prevent the over-division. Therefore, the reorganized state estimate (RSE), which is the transmitted data for sensor i, is expressed as follows: ˆxr i (t) = Hi(t)q( ˆxi(t)) (10) (10) where: : q( ˆxi(t)) =       q( ˆxi1(t)) q( ˆxi2(t)) ... q( ˆxin(t))       q( ˆxi(t)) =       q( ˆxi1(t)) q( ˆxi2(t)) ... q( ˆxin(t))       Hi(t) = diag{hi1(t), hi2(t), · · · , hin(t)}, hij(t) = ( 1, the jth component is sellected 0, others and Tr(Hi(t)) = ri(t) Here, for the jth components of sensor i, the median, the boundaries and the interceptive threshold for each component of state vector are given, and the parameters u, ρ, and Hi(t) need to be conﬁrmed. 4.2.2. The High-Accuracy Data Fusion Strategy 4.2.2. The High-Accuracy Data Fusion Strategy 4.2.2. The High-Accuracy Data Fusion Strategy In sensor node, the energy-efﬁcient data transmission strategy is executed and the RSEs are transmitted to the FC. Then, the high-accuracy data fusion strategy would be executed in FC. Firstly, each RSE ˆxr i (t) contains incomplete information of the original estimate ˆxi(t). Therefore, it is necessary to compensate each RSE for improving its estimated accuracy. In this case, the state estimate from the RSE of sensor i, denoted by ˆxc i (t), is proposed as: ˆxc i (t) = Hi(t)q( ˆxi(t)) + (I −Hi(t))A ˆxc i (t −1) (11) (11) where (I −Hi(t))A ˆxc i (t −1) is used to compensate for the components “0” of ˆxr i (t). It means that, if partial components of the ˆxi(t) are not transmitted to the FC at time t, they will be estimated by ˆxc i (t −1). i According to the previous process, the FC receives L (the number of sensors) state estimates ˆxc 1(t), ˆxc 2(t), . . . , ˆxc L(t). FC then fuses them to the ﬁnal estimate ˆx(t). In this paper, the weights of each sensor change over time t and the fusion estimate is given as follows: ˆx(t) = L ∑ i=1 Wi(t) ˆxc i (t) (12) (12) where Wi(t) = diag{wi1(t), wi2(t), . . . , win(t)}, and Σiwij(t) = 1. Thus, the ﬁnal fusion estimation can be calculated. The parameters of this model will be calculated in the next part. Sensors 2018, 18, 56 9 of 19 5.1. The Parameters Analysis in Energy-Efﬁcienct Data Transmission Strategy In energy-efﬁcient data transmission strategy, Hi(t), u, ρ and τ need to be conﬁrmed, where: Hi(t) = diag{hi1(t), hi2(t), · · · , hin(t)} (i = 1, 2, ..., L) u = [u1, u2, · · · , un]T, ρ = [ρ1, ρ2, · · · , ρn]T and τ = [τ1, τ2, · · · , τ L is the number of sensor nodes, and n is the number of components for the state vector. Here, uj, ρj and τj (j = 1, 2, . . . , n) are the ﬁxed parameters while Hi(t) (i = 1, 2, . . . , L) changes over time t. The parameters uj, ρj and τj come from the quantization method. Firstly, the minimum value of the MSE is calculated with some assumptions. 1. The jth component of the observed data is evenly distributed between sj + aj and sj −aj. 1. The jth component of the observed data is evenly distributed between sj + aj and sj −aj. h 1. The jth component of the observed data is evenly distributed between sj + aj and sj −aj. 2. The value sj + aj is located on one edge of the quantization method, where sj + aj = 1/2(1 + ρj)uj·ρjh 1. The jth component of the observed data is evenly distributed between sj + aj and sj −aj. 2. The value sj + aj is located on one edge of the quantization method, where sj + aj = 1/2(1 + ρj)uj·ρjh with a ﬁxed h (it is set to h = 1 for ease of calculation). 2. The value sj + aj is located on one edge of the quantization method, where sj + aj = 1/2(1 + ρj)uj·ρjh with a ﬁxed h (it is set to h = 1 for ease of calculation). 3. The value τj is located on other edge of the quantization method, where τj = 1/2(1 + ρj)uj·ρjk with a ﬁxed k (it is supposed k = k0). 3. The value τj is located on other edge of the quantization method, where τj = 1/2(1 + ρj)uj·ρjk with a ﬁxed k (it is supposed k = k0). 5. Parameters Analysis In last section, the MHEEFE model is proposed and two important strategies are introduced. However, some parameters should be conﬁrmed. Then the parameters in energy-efﬁcient data transmission strategy and high-accuracy data fusion strategy are analyzed respectively in this section. 5.1. The Parameters Analysis in Energy-Efﬁcienct Data Transmission Strategy 5.1. The Parameters Analysis in Energy-Efﬁcienct Data Transmission Strategy Then, the optimization Equation (15) should be discussed. When the parameters aj and k0 are fixed and assumed to 1 and 20, respectively. The function image of f is drawn approximately, and is shown in Figure 3. Figure 3. Function image of f with aj = 1 and k0 = 20. f Figure 3. Function image of f with aj = 1 and k0 = 20. Figure 3. Function image of f with aj = 1 and k0 = 20. Figure 3. Function image of f with aj = 1 and k0 = 20. In Figure 3, it is obvious that there is a minimum value of the function f with ρj ∈ (0, 1) when the parameters aj and k0 are fixed. However, f is a kind of higher-order polynomial function whose analytic solution of minimum value is hardly calculated. Therefore, an optimization method, such as newton descent method, is introduced to calculate the approximate solution. After calculating the parameters uj and ρj for all the components of the observed value, the In Figure 3, it is obvious that there is a minimum value of the function f with ρj ∈(0, 1) when the parameters aj and k0 are ﬁxed. However, f is a kind of higher-order polynomial function whose analytic solution of minimum value is hardly calculated. Therefore, an optimization method, such as newton descent method, is introduced to calculate the approximate solution. After calculating the parameters uj and ρj for all the components of the observed value, the parameter Hi(t) (i = 1,2, …, L) in dimensionality reduction method needs to be ascertained. Here, for sensor i, the parameter Hi(t) is used to make the MSE small enough. Thus, to satisfy the requirements above, an optimal problem is given as follows: ( ) [ ] t H T i After calculating the parameters uj and ρj for all the components of the observed value, the parameter Hi(t) (i = 1,2, . . . , L) in dimensionality reduction method needs to be ascertained. Here, for sensor i, the parameter Hi(t) is used to make the MSE small enough. 5.1. The Parameters Analysis in Energy-Efﬁcienct Data Transmission Strategy Then, we have: ( Then, we have: min τj,uj,ρj MSE xj, q xj (13) Then, we have: Then, we have: min τj,uj,ρj MSE xj, q xj (13) min τj,uj,ρj MSE xj, q xj (13) (13) where xj is evenly distributed between sj + aj and sj −aj. Therefore: where xj is evenly distributed between sj + aj and sj −aj. Therefore: where xj is evenly distributed between sj + aj and sj −aj. Therefore: MSE xj, q xj = k0 ∑ h=2 R sj+ 1+ρj 2 ·ujρjh−1 sj+ 1+ρj 2 ·ujρjh (sj+ujρjh−xj) 2dxj+ k0 ∑ h=2 R sj− 1+ρj 2 ·ujρjh−1 sj− 1+ρj 2 ·ujρjh (sj+ujρjh−xj) 2dxj 2(aj−τj) + R sj+τ sj−τ (sj−xj) 2dxj 2τj According to the symmetry of the quantization function, the MSE can be translated to: According to the symmetry of the quantization function, the MSE can be translated to: MSE xj, q xj = k0 ∑ h=2 R sj+ 1+ρj 2 ·ujρjh−1 sj+ 1+ρj 2 ·ujρjh (sj+ujρjh−xj) 2dxj aj−τj + 1 3τ3 j = (1−ρj) 3(1+ρj3) 24(aj−τj)(1−ρj3) u3 j ρ3 j −u3 j ρ3k0 j + 1 3τ3 j (14) (14) In assumptions 2 and 3: 1 + ρj ujρj 2 = aj, 1 + ρj ujρk0 j 2 = τj 1 + ρj ujρj 2 = aj, 1 + ρj ujρk0 j 2 = τj Next, combining Equation (14) and eliminating the parameters τj, an optimization formula is obtained: Next, combining Equation (14) and eliminating the parameters τj, an optimization formula is obtained: min aj,uj,ρj,τj MSE xj, q xj min aj,uj,ρj,τj MSE xj, q xj ⇒ min 0<ρj<1,aj,k0 f aj,ρj, k0 = a2 j 1+ρk0−1 j +ρ2(k0−1) j (1−ρj) 2 1−ρj+ρ2 j 3 1+ρj+ρ2 j (1+ρj) 2 + a3 j ρ3(k0−1) j 3 (15) aj,uj,ρj,τj ⇒ min 0<ρj<1,aj,k0 f aj,ρj, k0 = a2 j 1+ρk0−1 j +ρ2(k0−1) j (1−ρj) 2 1−ρj+ρ2 j 3 1+ρj+ρ2 j (1+ρj) 2 + a3 j ρ3(k0−1) j 3 (15) (15) 10 of 19 (15) 10 of 19 (15) Sensors 2018, 18, 56 m  Then, the optimization Equation (15) should be discussed. When the parameters aj and k0 are ﬁxed and assumed to 1 and 20, respectively. The function image of f is drawn approximately, and is shown in Figure 3. 5.1. The Parameters Analysis in Energy-Efﬁcienct Data Transmission Strategy Thus, to satisfy the requirements above, an optimal problem is given as follows: ( ) ( ) [ ] ( ) ( ) ( ) ( ) ( ) i i i i i i i t H t Ax t H I t x t H q t x MSE t s t H Tr i α ≤ − − + ⋅ )) 1 ( ) ( , ( .. min (16) t th ti (H (t) (t)) H (t) ( (t)) th ti l bl i t f d min Hi(t) Tr[Hi(t)] s.t. MSE(xi(t), q(Hi(t) · xi(t)) + (I −Hi(t))Axi(t −1)) ≤αi (16) (16) (16) According to the equation q(Hi(t)xi(t)) = Hi(t)q(xi(t)), the optimal problem is transformed as follows: According to the equation q(Hi(t)xi(t)) = Hi(t)q(xi(t)), the optimal problem is transformed as follows: According to the equation q(Hi(t)xi(t)) = Hi(t)q(xi(t)), the optimal problem is transformed as follows: ( ) [ ] t H T i According to the equation q(Hi(t)xi(t)) = Hi(t)q(xi(t)), the optimal problem is transformed as follows: i T [H (t)] ( ) ( ) [ ] i t H Tr min min ( ) Tr[Hi(t)] ( ) ( ) [ ] ( ) ( ) ( ) ( ) ( ) ( ) i i i i i i i t H t Ax t x q t H t Ax t x MSE t s t H Tr i α ≤ − − + − )1 ( )1 ( , .. min (17) min Hi(t) Tr[Hi(t)] s.t. where: where: cik = [xik(t) −akxi(t −1)]2 −[xik(t) −q(xik(t))]2 and q(xik(t)) is calculated according to Equation (9) ﬁnd the maximum in vector ci, which is check = max{ci} while check ≤threshold ﬁnd the maximum in vector ci, which is check = max{ci} ﬁnd the maximum in vector ci, which is check = max{ci} while check ≤threshold update the vector ci: delete max{ci} in ci; d t check check check + {c } update the vector ci: delete max{ci} in ci; update check: check = check + max{ci} update the vector ci: delete max{ci} in ci; update check: check = check + max{ci} update check: check = check + max{ci} end while 5.1. The Parameters Analysis in Energy-Efﬁcienct Data Transmission Strategy MSE(xi(t), Axi(t −1) + Hi(t)(q(xi(t)) −Axi(t −1))) ≤αi (17) (17) (17) On the other hand: On the other hand: ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) [ ] q p i i i i MSE MSE t Ax t x MSE t Ax t x q t H t Ax t x MSE − − − = − − − − )1 ( , )1 ( )1 ( , (18) MSE(xi(t), Ax(t −1) −Hi(t)(q(xi(t)) −Ax(t −1))) = MSE(xi(t), Ax(t −1)) − MSEp −MSEq (18) (18) (18) where: where: ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) t Ax t H t x t H MES MSE i i i p )1 ( , − = (19) MSEp = MES(Hi(t)xi(t), Hi(t)Ax(t −1)) MSEq = MSE(Hi(t)xi(t), Hi(t)q(xi(t))) (19) (19) and: and: ( ) ( ) ( ) ( ) ( ) ( ) t x q t H t x t H MSE MSE i i i i q , MESp −MSEq = 1 n n ∑ k=1 hk(t) h (xik(t) −akxi(t −1))2 −(xik(t) −q(xik(t)))2i (20) (20) Sensors 2018, 18, 56 11 of 19 where hk(t) is the sum of the kth row of matrix Hi(t), and ak is the kth row of matrix A. Therefore, the optimal problem becomes: where hk(t) is the sum of the kth row of matrix Hi(t), and ak is the kth row of matrix A. Therefore, the optimal problem becomes: min Hi(t) Tr[Hi(t)] s.t. n ∑ k=1 hk(t) h (xik(t) −akxi(t −1))2 −(xik(t) −q(xik(t)))2i ≥n(MSE(xi(t), Axi(t −1)) −αi) (21) min Hi(t) Tr[Hi(t)] s.t. n ∑ k=1 hk(t) h (xik(t) −akxi(t −1))2 −(xik(t) −q(xik(t)))2i ≥n(MSE(xi(t), Axi(t −1)) −αi) (21) min Hi(t) Tr[Hi(t)] (21) n ∑ k=1 hk(t) h (xik(t) −akxi(t −1))2 −(xik(t) −q(xik(t)))2i ≥n(MSE(xi(t), Axi(t −1)) −αi) Parameter Hi(t) can then be solved by an order-based algorithm. The computation procedures for the order-based algorithm are summarized in the order-based algorithm (Algorithm 1). Algorithm 1 Order-based Algorithm. For given s, τ, u, and ρ to determine the quantization function q(x), and for given the threshold αi. Description of some important values: Description of some important values: xi(t −1) is the last transmitted data saved by sensor i. xi(t −1) is the last transmitted data saved by sensor i. 5.1. The Parameters Analysis in Energy-Efﬁcienct Data Transmission Strategy xi(t −1) is the last transmitted data saved by sensor i. xi(t) is the estimated data in time t by sensor i. xi(t) is the estimated data in time t by sensor i. n is the dimensionality of xi(t). the dimensionality of xi(t). l h h h ld Calculate the threshold: Calculate the threshold: threshold = n(MSE(xi(t) −Axi(t −1)) −αi). Calculate the vector ci: Calculate the vector ci: ci = [ci1, ci2, · · · , cin] where: 5.2. The Parameters Analysis in High-Accuracy Data Fusion Strategy In the fusion estimation, FC receives only the compressed data from all sensor nodes, and fuses them with the parameter weights matrix. Normally, the weights should be related to the accuracy of each node. Here, the accuracy of compressed data with one node may change in a different time period. In addition, the accuracy of some sensor nodes may decline due to damage or failure. Therefore, the weights matrix changes over time t and is expressed as W(t). The computation procedures for the iterative method are summarized in the iterative method (Algorithm 2): 12 of 19 Sensors 2018, 18, 56 Algorithm 2 Iterative Method. For given CSEs of all sensors ˆxc 1(t), ˆxc 2(t), . . . , ˆxc L(t). Algorithm 2 Iterative Method. For given CSEs of all sensors ˆxc 1(t), ˆxc 2(t), . . . , ˆxc L(t). Initialization: Wi(1) = diag{1/L, 1/L, . . . , 1/L}, for i = 1, 2, . . . , L. FE(1) = 1 L ∑L i=1 ˆxc i (1), In time t (t > 1): for j = 1 to n if FEj(t −1) −ˆxij(t −1) = 0 w’ij(t) = n else w’ij(t) = 1 − \|FEj(t−1)−ˆxij(t−1)\| ∑n s=1\|FEs(t−1)−ˆxis(t−1)\|, for i = 1, 2, . . . , L and j = 1, 2, . . . , n. end if end for Normalize [w’1j(t), w’2j(t), . . . , w’Lj(t)] to become [w1j(t), w2j(t), . . . , wLj(t)], where Σiwij(t) = 1 Then, Wj(t) = diag{w1j(t), w2j(t), . . . , wLj(t)} And FEj(t) = L ∑ s=1 wsj(t) ˆxc sj(t) end for Normalize [w’1j(t), w’2j(t), . . . , w’Lj(t)] to become [w1j(t), w2j(t), . . . , wLj(t)], where Σiwij(t) = 1. Then, Wj(t) = diag{w1j(t), w2j(t), . . . , wLj(t)} L end for Normalize [w’1j(t), w’2j(t), . . . , w’Lj(t)] to become [w1j(t), w2j(t), . . . , wLj(t)], where Σiwij(t) = 1. Then, Wj(t) = diag{w1j(t), w2j(t), . . . , wLj(t)} L And FEj(t) = L ∑ s=1 wsj(t) ˆxc sj(t) 6. Simulation 6. Simulation In this part, two kinds of simulations: parameters simulation and performances simulation, are performed. The rate of convergence, the parameters k0 and threshold are simulated in the parameters simulation. The MSEs, communication trafﬁcs, and distribution of MSEs are simulated in the performance simulation. Meanwhile, for all simulations, the results are simulated 200 times, and the average of the 100 results is used as the ﬁnal simulation results. g Here, a dynamic system is used for simulation, whose state-space model is shown as follows: ( x(t + 1) = Ax(t) + B(ω(t) + v(t)) yi(t) = Cix(t) + Di(ω(t) + v(t)) ( x(t + 1) = Ax(t) + B(ω(t) + v(t)) yi(t) = Cix(t) + Di(ω(t) + v(t)) where ω(t) is the energy-bounded signal and v(t) is the white noise process with the initial state of 0. In parameters simulation, for more precise analysis, other parameters are simpliﬁed to reduce the effect on the analyzed parameter. Then, the system matrixes are: where ω(t) is the energy-bounded signal and v(t) is the white noise process with the initial state of 0. In parameters simulation, for more precise analysis, other parameters are simpliﬁed to reduce the effect on the analyzed parameter. Then, the system matrixes are: A =   0.9 0.1 0 0 0 0 0 0 0.9 0.1 0 0 0 0 0 0 0.9 0.1 0 0 0 0 0 0 0.9 0.1 0 0 0 0 0 0 0.9 0.1 0 0 0 0 0 0 0.9 0.1 0.1 0 0 0 0 0 0.9   , B = Di = 0.5I, Ci = I A =   0.9 0.1 0 0 0 0 0 0 0.9 0.1 0 0 0 0 0 0 0.9 0.1 0 0 0 0 0 0 0.9 0.1 0 0 0 0 0 0 0.9 0.1 0 0 0 0 0 0 0.9 0.1 0.1 0 0 0 0 0 0.9   , B = Di = 0.5I, Ci = I In performance simulation, a more practical system is needed. Therefore, an F-404 aircraft engine system is introduced and the system matrixes are: In performance simulation, a more practical system is needed. 6.1. Paremeters Simulation ignal is discussed. Figu t b f Firstly, the convergence of MHEEFE when the energy-bounded signal suddenly produces a large signal is discussed. Figure 4 shows the different iterations for the data convergence when the different number of components are transmitted, and compares with DMHFE model. nt number of components are transmitted, and compares with DMHFE model. s shown in Figure 4, the iterations of MHEEFE are always less than DMHFE for th ions. Here, it is assumed that nt is the number of transmitted components, and ndc is the As shown in Figure 4, the iterations of MHEEFE are always less than DMHFE for the same conditions. Here, it is assumed that nt is the number of transmitted components, and ndc is the number of drastically changed components. Then we have, when nt = ndc, the estimated value keeps a high accuracy. And the estimated value converges with 1 iteration step, when 2 × nt ≥ndc. Actually, if it is expected, the estimate converges after k (k ̸= 0) iteration steps; the minimum number of transmitted components should be [ndc/(k + 1)]. Therefore, we can obtain a sufﬁcient rate of convergence, as long as half of the state vector are transmitted in each period. stically changed components. Then we have, when nt = ndc, the estimated value keep cy. And the estimated value converges with 1 iteration step, when 2 × nt ≥ ndc. Actuall ed, the estimate converges after k (k ≠ 0) iteration steps; the minimum number of tran onents should be [ndc/(k + 1)]. Therefore, we can obtain a sufficient rate of convergence, of the state vector are transmitted in each period. econdly, Figure 5 simulates the averages and variances of MSEs and communication Secondly, Figure 5 simulates the averages and variances of MSEs and communication trafﬁcs with different k0. Here, the energy-bounded signal is not working, and the communication trafﬁcs are represented by the rate of actual trafﬁcs and maximum trafﬁcs. y g g ifferent k0. Here, the energy-bounded signal is not working, and the communication tra ented by the rate of actual traffics and maximum traffics. (a) (b) (c) (d) re 4. Iterations when the number of transmitted data changed: (a) The number of dr ged components is 1; (b) The number of drastically changed components is 3; (c) The nu ically changed components is 5; (d) The number of drastically changed components is 7 Iterations Iterations Iterations Iterations Figure 4. 6. Simulation Therefore, an F-404 aircraft engine system is introduced and the system matrixes are: A =   0.8673 0 0.2022 0.0293 0.9763 −0.0301 0.0259 0 0.8032  , B =   0.1 0.5 −0.2 0 0 0   It’s assumed two sensors collect the data. The related matrixes are: C1 = " 1 1 0 0 1 0 # , C2 = " 1 1 0 0 1 1 # , D1 = " 0.3 0.2 0.1 0.3 # and D2 = " 0.2 0.3 0.1 0.1 # Sensors 2018, 18, 56 remeters Simula 13 of 19 13 of 19 6.1. Paremeters Simulation ignal is discussed. Figu t b f Iterations when the number of transmitted data changed: (a) The number of drastically changed components is 1; (b) The number of drastically changed components is 3; (c) The number of drastically changed components is 5; (d) The number of drastically changed components is 7. (b) Iterations (a) Iterations (b) (a) (c) Iterations (d) Iterations (d) (c) 4. Iterations when the number of transmitted data changed: (a) The number of dr ed components is 1; (b) The number of drastically changed components is 3; (c) The nu ally changed components is 5; (d) The number of drastically changed components is 7 Figure 4. Iterations when the number of transmitted data changed: (a) The number of drastically changed components is 1; (b) The number of drastically changed components is 3; (c) The number of drastically changed components is 5; (d) The number of drastically changed components is 7. 14 of 19 er of Sensors 2018, 18, 56 changed com drastically ch Figure 5. The statistical data of MSEs and communication traffics with different k0. 0 10 20 30 40 50 60 70 80 90 100 k0 0 0.05 0.1 0.15 0.2 0.25 0.3 Average MSEs Variance MSEs Communication traffics Figure 5. The statistical data of MSEs and communication trafﬁcs with different k0. 18, 56 Figure 5. The statistical data of MSEs and communication traffics with different k0. Figure 5. The statistical data of MSEs and communication trafﬁcs with different k0. 8, 56 As shown in Figure 5, both the averages and variances of MSEs become stable with the increase of parameter k0. In fact, when k0 < 10, the proposed quantization only divides the range of true value into several subintervals. This division may lead to high and unstable MSE values. When k0 ≥10, this division can keep the MSE values lower and more stable. Besides, the communication trafﬁcs are still unstable when k0 < 20. It is means that when k0 stays in this range, the MHEEFE model needs to transmit more components to keep the MSEs stable. Therefore, when k0 is assigned to be 20 (or more than 20), the statistical data of MSEs and communication trafﬁcs can keep lower and more stable. As shown in Figure 5, both the averages and variances of MSEs become stable with the increase of parameter k0. 6.1. Paremeters Simulation ignal is discussed. Figu t b f In fact, when k0 < 10, the proposed quantization only divides the range of true value into several subintervals. This division may lead to high and unstable MSE values. When k0 ≥ 10, this division can keep the MSE values lower and more stable. Besides, the communication traffics are still unstable when k0 < 20. It is means that when k0 stays in this range, the MHEEFE model needs to transmit more components to keep the MSEs stable. Therefore, when k0 is assigned to be 20 (or more than 20), the statistical data of MSEs and communication traffics can keep lower and more stable. Finally like in Figure 5 the statistical data of MSEs and communication traffics with different Finally, like in Figure 5, the statistical data of MSEs and communication trafﬁcs with different threshold are simulated in Figure 6. As shown in Figure 6, the variances of MSEs show a little change when threshold changes, which means the MSEs remain stable all the time. On the other hand, the averages of MSEs show a great increase as the threshold increases. Moreover, when 0 < threshold < 0.1, the communication trafﬁcs show a dramatic decline and decreases gradually when threshold > 0.1. Therefore, if threshold is between 0.05 and 1.5 we can achieve good performance. Finally, like in Figure 5, the statistical data of MSEs and communication traffics with different threshold are simulated in Figure 6. As shown in Figure 6, the variances of MSEs show a little change when threshold changes, which means the MSEs remain stable all the time. On the other hand, the averages of MSEs show a great increase as the threshold increases. Moreover, when 0 < threshold < 0.1, the communication traffics show a dramatic decline and decreases gradually when threshold > 0.1. Therefore, if threshold is between 0.05 and 1.5 we can achieve good performance. Figure 6. The statistical data of MSEs and communication traffics with different threshold. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Thresholds 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Averages, Variances and Communication traffics Average MSEs Variance MSEs Communication traffics Figure 6. The statistical data of MSEs and communication trafﬁcs with different threshold. Figure 6. The statistical data of MSEs and communication traffics with different threshold. Figure 6. The statistical data of MSEs and communication trafﬁcs with different threshold. 6.2. Performance Simulation I thi t th fi l t 6.2. Performance Simulation Figure 7. The final estimations for three components with different models. 0 20 40 60 80 100 120 140 160 180 200 Iterations -1 -0.5 0 0.5 1st Ture Value DMHFE MHEEFE MHEEFE-QO 0 20 40 60 80 100 120 140 160 180 200 Iterations -5 0 5 10 2nd Ture Value DMHFE MHEEFE MHEEFE-QO 0 20 40 60 80 100 120 140 160 180 200 Iterations -1 -0.5 0 0.5 3rd Ture Value DMHFE MHEEFE MHEEFE-QO Figure 7. The ﬁnal estimations for three components with different models. Figure 7. The final estimations for three components with different models Figure 7. The ﬁnal estimations for three components with different models. Then, Figure 7 shows that all of fusion estimation models can achieve a good performance which is hardly to compare further. Therefore, some statistical results and communication traffic for each component with different models are calculated and shown in Table 2, where it can be seen that in both in the average and variance of MSEs, the MHEEFE-QO still shows lower values. However, the communication traffics for each component keep the highest values. Then, the DMHFE and MHEEFE are compared. In 1st component, DMHFE shows lower value both in the average and variance of MSEs while employing about 2.67 times of communication traffic in MHEEFE. In 2nd component, the MHEEFE shows lower value in statistical data while employing about 1.57 times of communication traffic in DMHFE. In 3rd component, DMHFE shows lower variance and similar average and communication traffic. Combining with the Figure 7, the true value in 1st and 3rd components are changed in a smaller range while changed in the larger range for 2nd component. In these components, MHEEFE still keeps in the acceptable level. In DMHFE, when the change range is small, it shows a good result. However, when the change range get larger, the average of MSEs turns Then, Figure 7 shows that all of fusion estimation models can achieve a good performance which is hardly to compare further. Therefore, some statistical results and communication trafﬁc for each component with different models are calculated and shown in Table 2, where it can be seen that in both in the average and variance of MSEs, the MHEEFE-QO still shows lower values. However, the communication trafﬁcs for each component keep the highest values. Then, the DMHFE and MHEEFE are compared. 6.2. Performance Simulation I thi t th fi l t 6.2. Performance Simulation In this part, the final estimations for three components with different models, which are DMHFE, MHEEFE and MHEEFE with Quantization only (MHEEFE-QO)), are compared firstly and shown in Figure 7. Then, the statistical data and communication traffic for each component with different models are calculated and listed in Table 2. After that, the distributions of MSEs for different models are simulated in Figure 8. Besides, Figure 9 simulates the MSEs comparison with the drastically changed components. And the comparison of transmitted data is simulated in Figure 10. Finally, the fault tolerances for different models are compared in Figure 11. In this part, the ﬁnal estimations for three components with different models, which are DMHFE, MHEEFE and MHEEFE with Quantization only (MHEEFE-QO)), are compared ﬁrstly and shown in Figure 7. Then, the statistical data and communication trafﬁc for each component with different models are calculated and listed in Table 2. After that, the distributions of MSEs for different models are simulated in Figure 8. Besides, Figure 9 simulates the MSEs comparison with the drastically changed components. And the comparison of transmitted data is simulated in Figure 10. Finally, the fault tolerances for different models are compared in Figure 11. 15 of 19 Sensors 2018, 18, 56 Table 2. Some statistical data and communication trafﬁcs for each component with different model Table 2. Some statistical data and communication trafﬁcs for each component with different models. Components Models Averages of MSEs Variances of MSEs Communication Trafﬁc 1st component DMHFE 0.0068 1.28 × 10−5 122.75 MHEEFE 0.0098 1.04 × 10−4 46 MHEEFE-QO 0.002 6.71 × 10−6 400 2nd component DMHFE 0.3081 0.0376 140 MHEEFE 0.0701 0.0102 220 MHEEFE-QO 0.0838 0.0172 400 3rd component DMHFE 0.0149 6.00 × 10−5 140.45 MHEEFE 0.0108 1.93 × 10−4 138 MHEEFE-QO 0.0042 2.97 × 10−5 400 nents Models Averages of MSEs Variances of MSEs Communication Trafﬁc In Figure 7, three ﬁnal estimations for three components are simulated to discuss the different performances of three models, respectively. DMHFE is the fusion estimation which is proposed in [16]. MHEEFE is the fusion estimation which is presented in this paper. MHEEFE-QO is the fusion estimation which only quantizes the data, and transmits the whole data to FC. Sensors 2018, 18, 56 15 of 19 MHEEFE is the fusion estimation which is presented in this paper. MHEEFE-QO is the fusion estimation which only quantizes the data, and transmits the whole data to FC. 6.2. Performance Simulation I thi t th fi l t 6.2. Performance Simulation In 1st component, DMHFE shows lower value both in the average and variance of MSEs while employing about 2.67 times of communication trafﬁc in MHEEFE. In 2nd component, the MHEEFE shows lower value in statistical data while employing about 1.57 times of communication trafﬁc in DMHFE. In 3rd component, DMHFE shows lower variance and similar average and communication trafﬁc. Combining with the Figure 7, the true value in 1st and 3rd components are changed in a smaller range while changed in the larger range for 2nd component. In these components, MHEEFE still keeps in the acceptable level. In DMHFE, when the change range Sensors 2018, 18, 56 16 of 19 is small, it shows a good result. However, when the change range get larger, the average of MSEs turns worse. As shown in Figure 8, the abscissa expresses the percentage of the maximum MSE value of three models. And in this ﬁgure, the values of MHEEFE and MHEEFE-QO are centralized between 0% and 20% of the maximum MSE, while the values of DMHFE are centralized between 0% and 50% of the maximum MSE. Obviously, MHEEFE and MHEEFE-QO gain the lower and more stable MSE than DMHFE. Sensors 2018, 18, 56 16 of 19 Sensors 2018, 18, 56 16 of 19 Figure 8. The distributions of MSEs with different models. Numbers of data Figure 8. The distributions of MSEs with different models. Figure 8. The distributions of MSEs with different models. Numbers of data Figure 8. The distributions of MSEs with different models. Figure 8. The distributions of MSEs with different models. Figure 8. The distributions of MSEs with different models. According to the Figures 7 and 8 and Table 2, MHEEFE model shows better performance on MSE, and transmits less data to FC. However, those figures and table are simulated without the drastically changed components. Figure 9 simulates the MSEs comparison with the drastically changed components. According to the Figures 7 and 8 and Table 2, MHEEFE model shows better performance on MSE, and transmits less data to FC. However, those ﬁgures and table are simulated without the drastically changed components. Figure 9 simulates the MSEs comparison with the drastically changed components. According to the Figures 7 and 8 and Table 2, MHEEFE model shows better performance on MSE, and transmits less data to FC. However, those figures and table are simulated without the drastically changed components. 6.2. Performance Simulation I thi t th fi l t 6.2. Performance Simulation Moreover, the MSEs go back to the normal level more quickly than DMHFE. The MHEEFE-QO always expresses better performance according to Figures 7–9 and Table 2. However, as shown in Figure 10, MHEEFE-QO shows the highest communication cost in three models. Moreover, communication cost in MHEEFE-QO is 3 times more than DMHFE, and about 6 times more is working. Because of the restricted of communication capacity, the increasing of MSE couldn’t be eliminated but weaken. Moreover, the MSEs go back to the normal level more quickly than DMHFE. g q y The MHEEFE-QO always expresses better performance according to Figures 7–9 and Table 2. However, as shown in Figure 10, MHEEFE-QO shows the highest communication cost in three models. Moreover, communication cost in MHEEFE-QO is 3 times more than DMHFE, and about 6 times more than MHEEFE. Obviously, MHEEFE-QO can hardly be used for energy constrained WBNs, and the comprehensive performance of MHEEFE is better than DMHFE. Sensors 2018, 18, 56 17 of 19 Sensors 2018, 18, 56 17 of 19 The MHEEFE-QO always expresses better performance according to Figures 7–9 and Table 2. However, as shown in Figure 10, MHEEFE-QO shows the highest communication cost in three models. Moreover, communication cost in MHEEFE-QO is 3 times more than DMHFE, and about 6 times more than MHEEFE. Obviously, MHEEFE-QO can hardly be used for energy constrained WBNs, and the comprehensive performance of MHEEFE is better than DMHFE. Sensors 2018, 18, 56 17 of 19 Sensors 2018, 18, 56 17 of 19 Figure 10. The sum of communication cost. Sum of communication cost Figure 10. The sum of communication cost. Figure 10. The sum of communication cost. Sum of communication cost Figure 10. The sum of communication cost. Figure 10. The sum of communication cost. Figure 10. The sum of communication cost. Finally, the MSEs comparison between the fixed weights and iteration-based weights is simulated. In this simulation, the number of sensor nodes needs 3 at least. Then, a new node should be added and the related matrixes are:     3 0 2 0 0 0 1 Finally, the MSEs comparison between the ﬁxed weights and iteration-based weights is simulated. In this simulation, the number of sensor nodes needs 3 at least. Then, a new node should be added and the related matrixes are: Finally, the MSEs comparison between the fixed weights and iteration-based weights is simulated. 6.2. Performance Simulation I thi t th fi l t 6.2. Performance Simulation In this simulation, the number of sensor nodes needs 3 at least. Then, a new node should be added and the related matrixes are:     =     = 2.0 5.0 3.0 2.0 and , 1 1 0 0 0 1 3 3 D C added sensor would be broken at the 50th iterati C3 = " 1 0 0 0 1 1 # , and D3 = " 0.2 0.3 0.5 0.2 #     =     = 2.0 5.0 3.0 2.0 and , 1 1 0 0 0 1 3 3 D C o eo e , e e a e se so ou be b o e a e 50 i e a io A a e a , i s oca estimated value always is [25, 25, 25]T. Then, the simulation is shown in Figure 11. Here, the MHEEFE and MHEEFE-QO use the iteration-based weights. Moreover, the new added sensor would be broken at the 50th iteration. And after that, its local estimated value always is [25, 25, 25]T. Then, the simulation is shown in Figure 11. Here, the MHEEFE and MHEEFE-QO use the iteration-based weights. Moreover, the new added sensor would be broken at the 50th iteration. And after that, its local estimated value always is [25, 25, 25]T. Then, the simulation is shown in Figure 11. Here, the MHEEFE and MHEEFE-QO use the iteration-based weights. Figure 11. MSEs with a broken node. As shown in Figure 11 when the 3rd node is broken and transmits false data all of model Figure 11. MSEs with a broken node. Figure 11. MSEs with a broken node. g th 3 d d i b k d t Figure 11. MSEs with a broken node. Figure 11. MSEs with a broken node. g , , great MSEs. As the iteration goes on, the MSEs of MHEEFE and MHEEFE-QO decrease to a low value like before, however, the MSEs of DMHFE shows a higher value than before and hardly decreases. Obviously, iteration-based weights provide a better fault tolerance than DMHFE. As shown in Figure 11, when the 3rd node is broken and transmits false data, all of models show great MSEs. 6.2. Performance Simulation I thi t th fi l t 6.2. Performance Simulation Figure 9 simulates the MSEs comparison with the drastically changed components. Figure 9. MSEs with the drastically changed components. A h i Fi 9 th MSE f th d l d At th 50th it ti th MSEs Figure 9. MSEs with the drastically changed components. MSEs Figure 9. MSEs with the drastically changed components. Figure 9. MSEs with the drastically changed components. Figure 9. MSEs with the drastically changed components. Figure 9. MSEs with the drastically changed components. As s o i igu e 9, e MSEs o ee o e s a e co pa e A e 50 i e a io , e e e gy bounded signal is working and great changes happen in state value. In DMHFE, there are no corresponding solutions. Then the MSE takes a tremendous increasing. In MHEEFE-QO, all of components are transmitted to FC all the time. Then the MSEs are hardly affected by energy-bounded signal. Finally in MHEEFE, when the energy-bounded signal is working, the corresponding strategy is working. Because of the restricted of communication capacity, the increasing of MSE couldn’t be eliminated but weaken Moreover the MSEs go back to the normal level more quickly than DMHFE As shown in Figure 9, the MSEs of three models are compared. At the 50th iteration, the energy- bounded signal is working and great changes happen in state value. In DMHFE, there are no corresponding solutions. Then the MSE takes a tremendous increasing. In MHEEFE-QO, all of components are transmitted to FC all the time. Then the MSEs are hardly affected by energy-bounded signal. Finally in MHEEFE, when the energy-bounded signal is working, the corresponding strategy is working. Because of the restricted of communication capacity, the increasing of MSE couldn’t be As shown in Figure 9, the MSEs of three models are compared. At the 50th iteration, the energy-bounded signal is working and great changes happen in state value. In DMHFE, there are no corresponding solutions. Then the MSE takes a tremendous increasing. In MHEEFE-QO, all of components are transmitted to FC all the time. Then the MSEs are hardly affected by energy-bounded signal. Finally in MHEEFE, when the energy-bounded signal is working, the corresponding strategy 17 of 19 17 of 19 Sensors 2018, 18, 56 is working. Because of the restricted of communication capacity, the increasing of MSE couldn’t be eliminated but weaken. 6.2. Performance Simulation I thi t th fi l t 6.2. Performance Simulation As the iteration goes on, the MSEs of MHEEFE and MHEEFE-QO decrease to a low value like before, however, the MSEs of DMHFE shows a higher value than before and hardly decreases. As shown in Figure 11, when the 3rd node is broken and transmits false data, all of models show great MSEs. As the iteration goes on, the MSEs of MHEEFE and MHEEFE-QO decrease to a low value Sensors 2018, 18, 56 18 of 19 like before, however, the MSEs of DMHFE shows a higher value than before and hardly decreases. Obviously, iteration-based weights provide a better fault tolerance than DMHFE. like before, however, the MSEs of DMHFE shows a higher value than before and hardly decreases. Obviously, iteration-based weights provide a better fault tolerance than DMHFE. 7. Conclusions This paper has investigated the mixed H2/H∞-based fusion estimation problem. Meanwhile, the energy-limited condition is considered. Then a novel mixed H2/H∞-based energy-efﬁcient fusion estimation model (MHEEFE) is proposed, presenting a dimensionality reduction-based data compression method. Unlike the existing dimensionality reduction method, the method in this model adopted the selective dimensionality reduction, which provides higher selective probability if it has a larger change. Furthermore, an iteration-based weight calculation algorithm is used in the FC to fuse the estimated received data. On the other hand, the parameters of mixed H2/H∞-based ﬁlter are calculated at the FC, and the sensor nodes only collect the data, choose the transmitted data, quantize the transmitted data and send them to FC. These operations work with less energy consumption and can be used by WBNs. Finally, the simulations discussed the appropriate dimension for transmitting to FC. Compared with other related models, the MHEEFE shows a better performance. Acknowledgments: This work was supported by the National Natural Science Foundation of China under G 1772064 and the Fundamental Research Funds for the Central Universities 2017YJS005. Author Contributions: Zhenjiang Zhang and Han-Chieh Chao conceived and designed the experiments; Chao Li performed the experiments; Chao Li analyzed the data; Zhenjiang Zhang contributed analysis tools; Chao Li wrote the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 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https://openalex.org/W2566840385	https://europepmc.org/articles/pmc5153695?pdf=render	English	null	“The way the country has been carved up by researchers”: ethics and power in north–south public health research	International journal for equity in health	2,016	cc-by	10,669	* Correspondence: aislingwalsh@rcsi.ie 1Department of Epidemiology and Public Health Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland Full list of author information is available at the end of the article © The Author(s). 2016 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: Despite the recognition of power as being central to health research collaborations between high income countries and low and middle income countries, there has been insufficient detailed analysis of power within these partnerships. The politics of research in the global south is often considered outside of the remit of research ethics. This article reports on an analysis of power in north–south public health research, using Zambia as a case study. Methods: Primary data were collected in 2011/2012, through 53 in-depth interviews with: Zambian researchers (n = 20), Zambian national stakeholders (n = 8) and northern researchers who had been involved in public health research collaborations involving Zambia and the global north (n = 25). Thematic analysis, utilising a situated ethics perspective, was undertaken using Nvivo 10. Results: Most interviewees perceived roles and relationships to be inequitable with power remaining with the north. Concepts from Bourdieu’s theory of Power and Practice highlight new aspects of research ethics: Results: Most interviewees perceived roles and relationships to be inequitable with power remaining with the north. Concepts from Bourdieu’s theory of Power and Practice highlight new aspects of research ethics: Northern and southern researchers perceive that different habituses exist, north and south - habituses of domination (northern) and subordination (Zambian) in relation to researcher relationships. Northern and southern researchers perceive that different habituses exist, north and south - habituses domination (northern) and subordination (Zambian) in relation to researcher relationships. Bourdieu’s hysteresis effect provides a possible explanation for why power differentials continue to exist. In some cases, new opportunities have arisen for Zambian researchers; however, they may not immediately recognise and grasp them Bourdieu’s hysteresis effect provides a possible explanation for why power differentials continue to exist. In some cases, new opportunities have arisen for Zambian researchers; however, they may not immediately recognise and grasp them. g g p Bourdieu’s concept of Capitals offers an explanation of how diverse resources are used to explain these powe imbalances, where northern researchers are often in possession of more economic, symbolic and social capita while Zambian researchers possess more cultural capital. g g p Bourdieu’s concept of Capitals offers an explanation of how diverse resources are used to explain these power imbalances, where northern researchers are often in possession of more economic, symbolic and social capital; while Zambian researchers possess more cultural capital. “The way the country has been carved up by researchers”: ethics and power in north– south public health research Aisling Walsh1, Ruairi Brugha1 and Elaine Byrne2 Aisling Walsh1, Ruairi Brugha1 and Elaine Byrne2 Walsh et al. International Journal for Equity in Health (2016) 15:204 DOI 10.1186/s12939-016-0488-4 Walsh et al. International Journal for Equity in Health (2016) 15:204 DOI 10.1186/s12939-016-0488-4 Open Access * Correspondence: aislingwalsh@rcsi.ie 1Department of Epidemiology and Public Health Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland Full list of author information is available at the end of the article © The Author(s). 2016 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. Background perspective: How does power influence international academic north–south public health research, and why do power imbalances exist and persist? Power for this paper is best defined as “the production, in and through social relations, of effects that shape the capacities of actors to determine their circumstances and fate [37]. While north–south dichotomies dominate the literature on north–south health research, the purpose of this paper is to highlight that a more nuanced approach to such dichotomies needs to be developed if we are to understand such partnerships. The value of international research collaborations has been well documented [1–5]. A review of the literature on health research between lower and middle income countries (LMIC) and higher income countries1 (HIC) - referred to in this paper as the ‘global north and south’ -confirms that power imbalances and inequities exist at each stage of the research process: from funding, to agenda setting, data collection, analysis and research outputs [6–12]. Some of these imbalances are structural (such as funding) [9, 10, 13–16] and some are related to inequitable relationships [11, 17, 18], though both are interlinked. Theoretical framework A north–south dichotomy dominates evaluations of north–south health research.2 Inequities and power imbal- ances between researchers in LMIC and HIC are often considered outside the immediate remit of research ethics, resulting in researchers, funders and institutions ignoring fundamental ethical issues of the politics and inequity of research in LMIC. Recently, there have been calls for con- sideration of the processes of collaboration between the north and south as ethical concerns [17–25]. This has been termed by some authors a ‘situated ethics’ of research: Concepts from Pierre Bourdieu’s theory of Power and Practice [38] provide a useful lens to structure and dis- cuss the situated ethics of research dimensions of public health and health systems research that emerged from this study. Bourdieu contends that power is at the heart of all social life [39] and hence are also at the heart of research relationships. At the centre of Bourdieu’s analysis is the question: why do social inequalities persist? Bourdieu’s theory [38] conceptualises action as the outcome of a relationship between Field, Habitus, and Capital. These concepts are useful in exploring the same question of inequalities in research relationships and are therefore examined in more detail here. “We are concerned that issues of ethics should be seen as integral to the whole research process… about researchers interrogating and responding to unequal power relations…” [20] Abstract Conclusions: Inequities and power imbalances need to be recognised and addressed in research partnerships. A situated ethics approach is central in understanding this relationship in north–south public health research. Conclusions: Inequities and power imbalances need to be recognised and addressed in research partnerships. A situated ethics approach is central in understanding this relationship in north–south public health research. Keywords: Zambia, Research partnerships, Situated research ethics, Power, Bourdieu Walsh et al. International Journal for Equity in Health (2016) 15:204 Page 2 of 11 Page 2 of 11 The field Bourdieu describes society as consisting of a system of fields, each one with its own structure which is semi- autonomous and set within a larger field of power [38]. Fields are structured spaces of dominant and subordin- ate positions that are organised around specific types of capital or combinations of capital; and where actors struggle to accumulate these different kinds of capital [40]. In the study described in this paper, the arena is the field of international academic north–south public health research. The occupants of positions within a field may be either agents (in this case researchers) or institutions (Universities or research institutions) which are constrained or enabled by the structure of the field [40]. This paper reports on an analysis of power in north– south public health and health systems research, specif- ically a ‘situated ethics’ of research analysis [11, 20, 25]. The focus on research partnerships is through the lens of researchers, who are the people that define, shape and execute such research studies, and include the broader issues of the politics and power of the research process, from agenda setting to capacity building, to authorship; and how research actors and institutions function and interact. For the most part, the literature does not desig- nate these as ethical issues, and most research ethics guidelines [26–28] and published studies of research ethics focus instead on traditional areas such as research ethics review processes [29–33] and informed consent [34–36]. However, we argue that these broader ethical issues are of equal importance. Research design g Qualitative data collection and analysis methods were selected for this study, to understand and discover southern and northern researchers’ interpretations and experiences of being involved in north–south health re- search collaborations [50, 51]. The lead author under- took 53 interviews (see Table 1). Twenty of these were Zambian researchers, and 8 were national level stake- holders who had been involved in setting up the Zambian health research system. Twenty five northern researchers were included in the sample, 4 of whom were considered to be north–south researchers: 3 of these were northern researchers who had lived in Zambia for a long period of time; and 1 South African researcher who was in- volved in a multi-country collaboration involving Zambia.3 Bourdieu contends that economic capital is “at the root of all other types of capital” [38] thereby proposing it as the capital with the most influence. Social capital refers to collaboration between individuals and groups. Bourdieu views social capital from the perspective of producing or reproducing inequality [38] rather than a way to promote equality. Bourdieu asserted that symbolic capital is a resource available to an individual based on prestige and recogni- tion [40]. Scientific capital is classed as a form of symbolic capital based on the prestige of the University they are attached to [44]. Table 1 Interviewee attributes Northern Zambian Total Geographic location North 21 - 21 Zambian - 28 28 North–south 4 - 4 Sex Male 11 18 29 Female 14 10 24 Institutional affiliation University 18 14 32 Research institution 3 8 11 Government - 3 3 NGO 4 3 7 Career level Junior-middle level 10 12 22 Senior 15 16 31 Background/training Biomedical 11 12 23 Social science 14 16 30 Table 1 Interviewee attributes Northern Zambian Total Cultural capital is identified by Bourdieu [38] as existing in three different states. Internalised: refers to dispositions that are internalised by the individual through socialisation and that constitute patterns of understanding, such as northern researcher or southern researcher culture. Objectified: referring to objects, such as books and scientific instruments that require specialised cultural abilities to use, such as the culture of science, specifically in health research. Institutionalised: the educational credential system, namely qualifications, degrees or titles [39], such as educational culture and work culture in north– south health research. According to Bourdieu, cultural capital is a major source of social inequality. Habitus Despite the recognition of power as being central to health research collaborations, there is limited, if any, detailed analysis of power within north–south health research. The published literature on such collabora- tions does not provide detailed insight into why power imbalances persist and where discussed are often from self-evaluations or reflections and as such may under- play inequalities in north–south health research. This paper addresses this gap from a situated ethics of research Habitus refers to the values and expectations of particular social groups that are acquired as a result of a long-term occupation in a social world [38]. Habitus defines what is possible within a certain group, generating a self-fulfilling prophecy [39]. Dominant and subordinate positions must be identified for all the participants in the field. Habitus has moments when it is out of phase, particu- larly when a field undergoes a transformation that changes its rules. A structural lag can occur in these Walsh et al. International Journal for Equity in Health (2016) 15:204 Walsh et al. International Journal for Equity in Health (2016) 15:204 Page 3 of 11 Page 3 of 11 Walsh et al. International Journal for Equity in Health (2016) 15:204 Methods Setting circumstances, −a ‘hysteresis effect’ [39] - between aspi- rations and changing opportunities. The concept of hysteresis is particularly useful for explaining northern and southern positions in health research, in particular for explaining why power imbalances exist and persist, i.e. why the status quo remains unchanged, despite an impetus for change at the global level, illustrated, for example, in the calls to build capacity for health re- search leaders in LMIC [41–43]. circumstances, −a ‘hysteresis effect’ [39] - between aspi- rations and changing opportunities. The concept of hysteresis is particularly useful for explaining northern and southern positions in health research, in particular for explaining why power imbalances exist and persist, i.e. why the status quo remains unchanged, despite an impetus for change at the global level, illustrated, for example, in the calls to build capacity for health re- search leaders in LMIC [41–43]. Zambia was chosen as a suitable single case study [45] that explores the ethics of health research between the global north and the global south. According to the Zambia Forum for Health Research, health research in Zambia is fragmented and underfunded [46]; health re- search priority setting has been ad hoc [47] and external donors fund up to 90% of health research [48]. Recent years have witnessed attempts to establish a national health research system, including legislation and regula- tion [49]. In addition two of the authors have undertaken health research in Zambia over a number of years and therefore have knowledge of the context. Capital s draw upon e their posi- people accu- Fields are social, sym- mulation of ctory and in north–south l is “at the y proposing ocial capital and groups. rspective of ather than a s a resource and recogni- of symbolic sity they are 8] as existing re cialisation nding, such rcher books and lised tion [49]. In addition two health research in Zambi therefore have knowledge o Research design Qualitative data collectio selected for this study, southern and northern re experiences of being invo search collaborations [50 took 53 interviews (see T Zambian researchers, and holders who had been invo health research system. Tw were included in the samp to be north–south researc researchers who had live of time; and 1 South Af volved in a multi-country c Table 1 Interviewee attribute N Geographic location North 2 Zambian - North–south 4 Sex Male 1 Female 14 Institutional affiliation University 18 According to Bourdieu, individuals and groups draw upon a variety of resources to maintain and enhance their posi- tions in the social order [39]. The capital that people accu- mulate defines their social trajectory [44]. Fields are organised around a combination of economic, social, sym- bolic and cultural capital. Researchers’ accumulation of capital can help us to understand their trajectory and in particular power differentials in the field of north–south health research. Research design He asserted that sharing similar forms of cultural capital with others creates a sense of collective identity and group position [40]. Page 4 of 11 Page 4 of 11 Walsh et al. International Journal for Equity in Health (2016) 15:204 A purposive sampling strategy was employed, which in- volved selecting participants on the basis of their academic background, gender, roles and career stage/experiences in north–south health research. working, and by the University of Zambia Humanities and Social Sciences Research Ethics Committee. Thematic analysis [54] was undertaken using Nvivo 10. Reflexivity was central to the research process given the nature of the research, ie the primary researcher was analysing the field of north–south health research, but is also an actor (northern researcher) within the field. In keeping with the spirit of ethical north–south health research, a Zambian collaborator was invited to work with the northern researcher. The collaborator assisted with setting up the interviews, and provided contextual and cultural understanding. Sampling was conducted by undertaking a systematic mapping exercise of Zambian and northern health re- searchers. Mapping included: researcher, institution, and project/collaboration, years of project/collaboration, topic, discipline(s) career stage. Initially, the intention of the au- thors was to identify a broad range of southern (Zambian) researchers who had participated in health policy and systems research partnerships, excluding clinical research collaborations. However, it became clear that most re- searchers who fell within this category identified their re- search as lying within the broad field of public health research. The primary inclusion criterion was public health researchers involved or recently involved (up to 5 years ago) in academic public health research involving Zambia and a northern country. Researchers were ex- cluded where they had been involved in north–south health research studies that were operational for less than one year at the time of sampling, and where eligible re- search studies had been completed more than five years prior to the commencement of data collection for this study. The lead investigator sought to recruit a balance of senior (more experienced) and junior (less experienced) respondents. Hence, the respondents were selected based on their characteristics, in line with Given’s definition of purposive sampling [52]; and to obtain information and insights from those especially knowledgeable about or ex- perienced with north-south partnerships [53]. The field: north–south health research A plethora of guidelines exist for north–south health research collaborations [4, 5, 46, 55, 56]. For the most part, these rules of research collaborations were broadly adhered to in the collaborations explored in this study, including for example the principles of honesty, ac- countability, professional courtesy, fairness and good stewardship, as outlined in the Montreal Statement on Research Integrity (2013). However, only one out of 14 health research collaborations in this study had developed ethical guidelines relating to partnership governance. Habitus of researchers d ’ f International Journal for Equity in Health (2016) 15:204 Walsh et al. International Journal for Equity in Health (2016) 15:204 to collaborate better with northern researchers than devel- oping capacity for independent country-level research: to collaborate better with northern researchers than devel- oping capacity for independent country-level research: However, some northern researchers noted that cau- tion should be displayed against placing the onus on Zambian researchers to be the sole agents of change, instead recognising that northern researchers have a role to play in assisting to strengthen the capacity of Zambian researchers, to enable them to avail of these opportunities. “I think one has to look at the history and say at what point and how do you do capacity building that’s not patronising in nature, and it’s not just simply about building up people’s capacity to collaborate better with Western researchers so that we get better data (north–south researcher 1). It was reported by some researchers—north and south—that some northern researchers have an interest in maintaining the status quo, in terms of maintaining control over the research process. However, findings show that northern researcher motivations in many cases are altruistic, in terms of improving health in Zambia, and capacity building for local researchers, or illustrate a combination of securing career opportunities. North–south inequities were verbalised throughout the interviews by northern and southern researchers, alike. Even when multiple identities of northern and southern researchers and a shared culture of research were men- tioned by some interviewees, it was usually in addition to, rather than in the place of, north–south imbalances. It was clear that the northern/southern habitus was deeply rooted in these relationships. For example, in some situa- tions, Zambian researchers considered it ‘natural’ for northern researchers to set the agenda. There was often an assumption by Zambian researchers that research questions and data collection tools would be drafted in the north and adapted to the Zambian context, even in studies that were being undertaken in Zambia alone. This illustrates an acceptance of the status quo, in that this is how research partnerships have always operated. A number of northern researchers explained that active attempts to address power imbalances and inequities were not successful. An example was given where a northern research institution played a non-interventionist role in one north–south health research collaboration, because they did not want to be seen as the dominant partner. Habitus of researchers d ’ f However, this laissez-faire approach led to a perception that they were not pulling their weight on the project. A number of northern researchers stated that the culture of the research institution in Zambia remains colonial, instilling in Zambians a culture of taking the back seat in north–south health research. In addition, some Zambian researchers accused northern researchers of continuing to carve up the country for research studies, with post-colonial connotations. Despite the acceptance of the status quo, many inter- viewees considered these imbalances to be unreasonable. Some examples were given by northern interviewees where opportunities to change the status quo had arisen for Zambian researchers: namely, opportunities to input into the research agenda, to lead data analysis and to lead on authorship of journal articles. However, some inter- viewees suggested that in many cases these opportunities had not yet been taken up, or that change was occurring at a slow pace. “The country has been carved up by researchers and you stay in a guest house and you’re aware that XYZ universities in the US and UK are kind of ‘oh what are you working on?’ and there is almost a cautiousness about, oh well stepping on someone else’s feet, and that is so incredibly omnipresent in Zambia. And so there is this sense of too many researchers.” (northern researcher 13) Bourdieu’s hysteresis effect can provide a possible ex- planation for why this was the case. Perhaps these changes in north–south health research were subtle and the new emerging opportunities for Zambian researchers to lead had not yet been taken on board by many of them. There were also reports that Zambians often did not have their own research agenda prepared. Habitus of researchers d ’ f Bourdieu’s concept of habitus provides a valuable tool to gain insight into why north–south inequities and power imbalances exist and are perpetuated in north– south health research collaborations. Different habituses (experiences and expectations), constituting a dichotomy between north and south, exist in north–south research partnerships. The legacy of colonialism was mentioned many times - equally by Zambian and northern re- searchers. Some northern and southern interviewees linked different work practices and approaches, by northern and southern researchers, to the culture of aid and colonisation. This association was sometimes attrib- uted to Zambia having received considerable levels of aid contributing to the expectation that northern researchers would come to solve problems. Fourteen research collaborations were identified from the researchers selected. Sampling of researchers to interview from these collaborations included both a northern and southern representative from each of these collaborations. Six of these were classified as health policy and systems research collaborations, while 8 were in the broader field of public health, and 3 of which were Randomised Controlled Trials. Eight of these collaborations were multi-country research stud- ies involving more than one northern and more than one southern country. Six were bilateral studies be- tween Zambia and a northern country. A number of Zambian researchers believed that Zambian researchers themselves had accepted these in- equities and had chosen to work within the confines of the structure of what was presented to them. For example it was frequently mentioned that they accepted mid-level research positions, without striving for leadership positions, leaving these to their northern counterparts. Zambian respondents were frequently frustrated that so-called cap- acity building was in reality ‘exposure’. For some, this was considered to be patronising, as the form of capacity being developed was more about enabling Zambian researchers In-depth interviews were conducted between February and December 2011. Topic guides were developed which included researcher’s experiences of north–south health research at the various stages of the research process. Twenty six of the 28 Zambian interviews were con- ducted face to face, with 2 taking place over the phone. Five of the 25 northern interviews were conducted face to face and the remainder over the phone. Interviews were recorded and transcribed. Ethical approval was granted by the Royal College of Surgeons in Ireland Research Ethics Committee, where the lead author was Page 5 of 11 Walsh et al. International Journal for Equity in Health (2016) 15:204 Walsh et al. Social capital l Social capital is particularly valuable in analysing re- searcher relationships in north–south research collab- orations. While northern researchers generally had more direct access to social capital, due to connections with donors, both northern and southern partners mentioned being dependent on one another. This is due to donor requirements to include certain countries in a research bid, because the focus of the research is in the south, but also because the National Health Research Act (2013) stipulates that a Zambian must be a Principal Investigator or co-Principal Investigator on every study. A common perception existed, by Zambian researchers, of poor individual Zambian researchers versus affluent individual northern researchers, and the oft repeated belief that Zambian researchers choose a research car- eer only for the salary. It was frequently considered by Zambian researchers that choice due to actual interest in research or a particular research topic, was some- times seen to be a northern luxury. One of the reasons given by Zambian researchers as to why research part- nerships with the north should continue in the future was high northern economic status, which would provide opportunities for Zambians as well as northern researchers. It was also hinted by some northern researchers that they have too much to lose (i.e. their careers) by relinquishing control over funding. Examples were given by both southern and northern re- searchers that the latter used connections with Zambian researchers to steamroll agendas that northerners wished to pursue: “They needed the African data to push their agenda forward. So in a way it was almost contract research. It was really their study idea but our study site” (north–south researcher 23). However some Zambian researchers considered that the onus is partly on Zambian researchers, to seek out northern re- searchers who have an interest in health research topics of relevance in Zambia. However, eligibility for funding is often pre-defined, where northern researchers have ring fenced access to their own national or EU research funding for pre-defined topics, and is therefore outside the control of Zambian researchers. A number of Zambian interviewees stated that Zambian researchers could set the agenda, and that they themselves only accepted a proposal if it matched Zambian priorities. Capitals: north–south distribution Economic capital For the collaborations sampled, research funds flowed almost exclusively – with the exception of one study where funds were routed through a South African partner–through northern institutions, thereby instilling economic cap- ital with the northern partners .4 Indeed, the primacy of economic capital was recognised by researchers them- selves, many of whom concluded that as long as funding flows solely through the north, this will ensure that power remains with the north, no matter how much possession of other capitals shifts to either a state of equilibrium, or in favour of Zambian researchers. “When you are constrained financially, I don’t know whether it is because of poverty, there comes a point where you stop thinking what you can do. … And so it is only when they actually see the money coming in that is when they start to think. We find computers with dust on them, we have vehicles that have been used but have not been used for any research. So in that sense you can blame us as the southern, the poor people, in not having enough of this stimulus on our own…” (Zambian researcher 5) Page 6 of 11 Walsh et al. International Journal for Equity in Health (2016) 15:204 Walsh et al. International Journal for Equity in Health (2016) 15:204 Interviewees repeatedly cited research donors and northern researchers as occupying the dominant pos- ition in terms of dictating both the broad agenda and the partnership format, showing that being in posses- sion of economic capital secures a knock-on effect for control in other elements of the research process. Many interviewees, north and south, pointed to the inability or failure of Zambian researchers to directly access re- search grants, and also noted that this was mirrored in Zambian research institutions, which – according to some respondents–lacked economic capital, through an unwillingness of donors to support them in building institutional capacity. who provide comfort that the money will be used properly for the research that it was intended for” (Zambian researcher 3). Zambian Research Ethics Committees (RECs) were sometimes perceived by both northern and Zambian re- searchers to rubber stamp REC authorisation to obtain international funding. This was seen as unethical by some northern researchers. Social capital l “We have to look at ourselves as Western researchers as to ‘what are we doing here?’ We can’t deny, as far as I’m concerned, the negative things that we are doing, and also what we need to give away. I mean if you’re actually looking for equity and balance, somebody’s got to give away something, and it’s pretty clear who has to give away stuff.” (northern researcher 5) Zambian researchers sometimes viewed that they ‘sell themselves’, accepting northern initiated research part- nerships in order to increase their economic capital, even though the agenda did not accord with Zambian priorities. A perspective that strongly emerged from northern and southern researchers was that even where there are positive social connections between researchers, such as trust and respect, north–south inequities and power imbal- ances continued to exist due to imbalances in economic capital. One perspective, reported by both Zambian and northern researchers, was that donors did not trust southern researchers to have the capacity to manage funds and account for the research budget, instead pla- cing more trust in northern partners. It was considered by some northern and Zambian researchers that pla- cing money in the hands of northern partners, gives do- nors an element of security that funding will be utilised in the most effective manner, making it easier to hold them to account for spending. This was described as important for the donors, “to be assured that there is some amount of eyes and ears from northern partners, Cultural capital l l l One South African researcher reported being ‘forced’ to undertake research analyses, i.e. did so reluctantly, even when she had not been involved in the design of the study and lacked knowledge of the Zambian health system. Through an analysis of Bourdieu, this can be at- tributed to symbolic/scientific capital of northern and South African researchers, through an assumption that their capacity will be higher. Cultural capital plays a major role in research partnerships that transcend cultural and geographic divides. Despite interviewees’ views that northern researchers lacked un- derstanding of context in agenda setting and research design, a number of Zambian interviewees recounted situations where northern researchers insisted on stan- dardised questions, even though they were not well suited to the Zambian context. “I had no input in the research design, how the questionnaires were developed, how the research was conducted, and then this mythical idea that I could come in and I could just look at the data and write-up, so really it was completely ridiculous. It still perplexes me as to how people thought that I could just look at data and come in, having not been at all involved, and from South Africa.” (north–south researcher 17) A number of northern and Zambian researchers men- tioned that the culture of the Zambian research institution is colonial (institutionalised cultural capital), which instils in Zambians the practice of taking a back seat in health re- search. Both Zambian and northern researchers described that northern researchers lead on data analysis, even though in most cases they did not lead on the data collec- tion process and were often perceived to have a poor un- derstanding of Zambian contextual issues. Some northern researchers were acutely aware of this, expressing their discomfort with the situation. However, other northern researchers did not see this deficiency in cultural aware- ness as presenting a problem. This suggests that symbolic/ scientific capital of northern researchers can override the cultural capital of those in the south, through their as- sumption that they have greater capacity to undertake data analysis. Published literature reveals that most articles relating to LMIC contain authors from HIC and also that the first author is likely to be from a HIC [10, 58, 59]. This study found evidence on inequities in author- ship of papers from northern and southern perspectives. Symbolic and scientific capital The perception existed by many northern and Zambian interviewees, that Zambian research institutions did not have the capacity to manage funds and were seen by do- nors to be risky. This was considered to be in contrast with northern researchers/ institutions possessing the prestige to be viewed by funders as trustworthy. This Walsh et al. International Journal for Equity in Health (2016) 15:204 Page 7 of 11 Page 7 of 11 “So now I’m trying to submit a paper on his behalf, he’s the first author. I can’t even get hold of him to get him to agree that it’s ok, it can go in. But ultimately if there is a problem with it he’s the first author. And I’m not sure what the ethics of…should I submit a paper with somebody else’s name first? But if I put my name first that would be wrong.... It doesn’t seem quite right but I suppose maybe that’s the way these collaborations do work in the end.” (northern researcher 8) could be attributed to the reputation—or symbolic cap- ital and lack of it—which both north and Zambians have acquired over time. It could also be based on genuine reasons for considering Zambian researchers (or their institutions) as risky. y There were examples cited of scientific capital at play. In one case it was reported that two northern re- searchers had pursued a particular research topic, des- pite the Zambian Ministry of Health stating that it was not relevant to the needs of the country. This suggests that scientific capital or ‘academic power’ [57] stemmed from or was reinforced by the economic capital of the northern researcher. Other examples included northern researchers reporting a general Zambian unwillingness to input into drafts of research proposals. This could signify an absence of ownership of the research; and/or a lack of capacity to input, both of which can be inter- preted as less (or less confidence in their) scientific capital. However, other researchers stated that it is the norm for the first and senior author to be non-Zambians and for the Zambians to be in the middle. Most respondents discussed research capacity strength- ening from a one-way, north-south perspective, suggest- ing that due to the symbolic (scientific) capital of northern researchers, there is an assumption that they alone have the knowledge and capacity to impart to the south. Cultural capital l l l Despite some interviewees indicating that the process was seen as equitable and ethical with both northern and Zambian researchers having equal opportunities to publish, several experiences of authorship being contro- versial were communicated. Many northern interviewees reported a bias in favour of including Zambian collabo- rators, claiming that even though northerners undertook most of the writing, they could not put themselves as first author. Some researchers, north and south, saw this as being unethical, whereas other perceived it as being important in terms of visibility and building capacity for southern researchers. At the point of fieldwork, Zambian respondents often viewed northern researchers as lacking the capacity to understand the culture and context of communities, particularly those who participated in fieldwork for only short periods of time. Northern researchers were often aware that their presence could have an influence on the research, such as an expectation that participation in research would result in an increase in health ser- vices. Many Zambian researchers also shared this view. Page 8 of 11 Page 8 of 11 Walsh et al. International Journal for Equity in Health (2016) 15:204 Walsh et al. International Journal for Equity in Health (2016) 15:204 Walsh et al. International Journal for Equity in Health (2016) 15:204 “They will say ‘oh here is a muzungu, this woman what does she want, let’s tell her what will make her happy’. So much of the time the way they will answer will not be because of what they think, but they will say what is it that she wants to be happy with. So they will try to please you.” (Zambian researcher 14) stages of the research process. Firstly, habitus helps us to understand the sometimes unconscious maintenance of the status-quo in north–south health research, which most southern and many northern researchers see as inequitable. This article illustrates that the views of both sets of researchers support the conclusion that differ- ent habituses exist, north and south, which are central to understanding the dynamics and ethics of north–south health research. These are habituses of domination (northern) and subordination (Zambian) in relation to re- searcher relationships. It is recognised that these are gen- eralisations and stereotypes, used for typing and analysis of findings. However, the results showed a propensity among most respondents from each location to gravitate towards these states. Cultural capital l l l Some northern respondents felt that it was important that they take part in some of the data collection - recognising the need to acquire some degree of cultural capital. A number of Zambian interviewees contended that Zambian researchers need to be “custodians of our own culture” (Zambian researcher 21), suggesting they should have an innate closeness with the communities being researched, even when they were not considered to be complete insiders, and sometimes quite distanced from the communities of study. Secondly, Bourdieu’s hysteresis effect provides a pos- sible explanation for why power differentials continue to exist. In some cases, new opportunities have arisen for Zambian researchers; however, they may not imme- diately recognise and grasp them [44]. Perhaps these changes in north–south health research are subtle and the increasing opportunities for Zambian researchers (in agenda setting, data analysis and authorship) have not yet been fully recognised or taken on board by the Zambian researchers. Studies in the area of north– south health research consistently report a lack of cap- acity in the south as being one of the major reasons for north–south power imbalances [41, 42]. It may also be the case that research donor claims that they wish to see southern-driven research has been lip-service, not supported by institutional capacity-building action. If this is the case, new opportunities without sufficient capacity strengthening will result in the maintenance of the status quo. “Sometimes…I am corrupted too. When I was growing up… I knew my own language very well. But then as you grow up and you begin to go into English… I lose track… But somehow I believe I am one of them. But I need to respect and understand my people better because I have been out for a long time. We as educated Zambian researchers should understand that we have gaps in trying to reach and understand our own communities as well.” (Zambian researcher 17) Discussion While a number of researchers spoke of trust and equity amongst partners, many perceived relationships and roles to be inequitable with power remaining in the north. An analysis of findings has shown that northern researchers were more likely than Zambian researchers to consider power imbalances as ethical issues, with Zambian researchers more likely to consider as ethical issues, traditional research ethical concerns, such as re- search ethics review processes and informed consent. q Thirdly, Bourdieu’s’ concept of Capitals allows us to explain how diverse resources are used to explain these power imbalances, where northern researchers are often in possession of more economic, symbolic and social capital vis-a-vis Zambian researchers; while Zambian researchers possess more cultural capital vis-a-vis northern researchers. Literature in the area of inter- national health research collaborations illustrates that while power is regularly reported in north–south health research [6, 7, 18, 25], it is rarely discussed in detail be- tween research partners. Recognition of the different di- mensions and fields of power that are occupied and exercised by northern and southern researchers could pave the way for more equitable partnerships. For ex- ample, by explicitly recognising and valuing the different forms of capital, greater weight would be given to non- economic capital and recognition of the contribution of all partners. This could help not only achieve greater north–south equity, but could also lead to more rigorous and more culturally contextualised research. This paper This study highlights the perspectives of one set of actors—researchers—in respect to the different dimen- sions and different distributions of powers in research col- laborations, through concepts from Bourdieu’s theory of Power and Practice. Though not considered in this paper, it is recognised that other actors, such as research donors, research participants, communities and policy makers are also key players to be considered. Furthermore, dissemin- ation and getting research into policy and practice stages of the research process have not been included, which would be desirable to gain a complete picture of power across each stage of the research process. Both of these were beyond the scope of the study conducted. The application of concepts from Bourdieu’s theory of Power and Practice [38] has a number of uses for highlighting new aspects of research ethics at different Walsh et al. Conclusions This article has highlighted the importance of consider- ing as ethical issues, not just traditional notions of re- search ethics, such as research ethics review processes but also macro research ethics, of which power is a cen- tral component of international public health research involving the global north and the global south. The argument that power imbalances need to be redressed is based on an assumption that research part- nerships should at the very least balance knowledge, interest and power in the short term, with the aim of maximising benefits for LMICs in the longer term. Many of the imbalances are structural, rather than within the realms of researcher relationships. This means that even if relationships can be altered, struc- tural inequities will remain dominant (for example eco- nomic capital in the form of research funding), thereby ‘trumping’ symbolic, social or cultural capital. Address- ing relationship inequality is often constrained by in- equalities at the structural level, which is within the hands of other actors in the research process, such as donors. Discussion International Journal for Equity in Health (2016) 15:204 Page 9 of 11 Page 9 of 11 term; and there was a consensus among northern and southern researchers, broadly speaking, that the mutual benefits outweighed the disadvantages of north–south collaborations. However, inequities and power imbalances need to be recognised and addressed and the situated eth- ics approach, taken in this paper, needs to be seen as a central ethical concern in public health research. therefore encourages the scope of ethical reflection to be broadened to consider the broader situated ethics of north–south health research, which “takes into account the realities of complex individual, institutional and national imbalances in power and resources.” [11] Bourdieu’s work has been criticised for concentrating on the internal analysis of fields, which may encourage a loss of sight as to how fields are connected into broader society; and his framework stresses the propen- sity to perpetuate structures inherited from the past, ra- ther than encouraging researchers to seek out forms of change [57]. This study has used Bourdieu’s analytical framework to explore and understand the views, ac- tions and non-actions of researchers interviewed rather than to understand how the situation could change. p This concept of situated ethics has potential relevance and application to southern Africa, for example in un- derstanding power differentials in research relationships between southern Africa and HIC. A recognition of the different elements of power by northern and southern researchers could pave the way for more equitable part- nerships. This can be achieved through recognising the importance of considering as ethical issues, not just trad- itional notions of research ethics, but also a situated re- search ethics, as described in this paper. Considering only one of the partnerships in this study had developed re- search ethics guidelines relating to the partnerships them- selves, it is recommended that research partnerships incorporate a situated ethics approach by developing ethical guidelines relating to health research partnership governance and for operationalising these, or; contextually adapting and utilising existing health research partnership guidelines such as the Swiss Commission KFPE Guide for Transboundary Research Partnerships (2012) [4] Endnotes 1A systematic search process identified 8,000 potential references, 239 of which fitted the inclusion criteria. An abundance of commentaries and debates exist, and where empirical research exists, it is overwhelmingly in the form of self-reflection and self-evaluation which usually focus on one aspect of the research partnership or research process, for example research capacity strengthening or authorship in north–south health research. Ninety-four empirical studies were identified, 57 relating to macro re- search issues, and 37 relating to micro research ethical issues. The literature review identified 13 evaluations of north–south health research, of which 9 were self- evaluations. Following piloting of search terms relating broadly to north–south health research, 4 databases were systematically searched: Web of Knowledge, Pubmed, Global Health, and Scirus. The following search terms/ strings were used: (health OR medical OR medicine OR biomedical OR biomedicine OR clinical OR “tropical medicine”) AND (partner* OR network* OR consort* OR collaborate* OR alliance* OR cooperate* OR co-operate) AND (research) AND ((global OR international OR trans- national OR multi-country OR (multi AND country) OR cross-country OR (cross AND country) OR (north AND south)) OR (“developing country” OR “developing coun- tries”) OR ((lower OR middle) AND income)). The follow- ing inclusion criteria were applied. To date, most studies of north–south health research collaborations have been self-evaluations. While this in itself is positive, often authors do not state their posi- tionality in relation to the research. Therefore, it is im- portant to explicitly acknowledge that the three authors of this paper are all northern (Irish) public health / health systems researchers, even if two of them have lived and worked for six and twenty years, respectively, in sub-Saharan Africa and the other has worked and visited Zambia as part of one research partnership. The use of concepts from Bourdieu’s theory of Power and Practice could be used to explain power differentials in other forms of partnership between northern and other southern countries. The findings in this paper support the view that the future for north-south health research is one of co-dependency, at least in the short to medium Page 10 of 11 Walsh et al. International Journal for Equity in Health (2016) 15:204 1. Research on: human health/ (bio)medicine/clinical/ tropical disease. Received: 12 August 2016 Accepted: 28 November 2016 Received: 12 August 2016 Accepted: 28 November 2016 Funding Irish Aid partly funded this study, through the Global HIV/AIDS Initiatives Network. 16. Davey S. The 10/90 report on health research 2003–2004, in The 10/90 Report on Health Research 2003–2004. Geneva: Global Forum for Health Research; 2004. p. xxvi + 282. 16. Davey S. The 10/90 report on health research 2003–2004, in The 10/90 Report on Health Research 2003–2004. Geneva: Global Forum for Health Research; 2004. p. xxvi + 282. Abbreviations HIC: High income country; LMIC: Low and middle income country; REC: Research Ethics Committee 12. Crane J. Adverse events and placebo effects: African scientists, HIV, and ethics in the ’global health sciences’. Soc Stud Sci. 2010;40(6):843–70. 12. Crane J. Adverse events and placebo effects: African scientists, HIV, and ethics in the ’global health sciences’. Soc Stud Sci. 2010;40(6):843–70. 13. Ijsselmuiden CB, et al. Evolving values in ethics and global health research. Glob Public Health. 2010;5(2):154–63. 13. Ijsselmuiden CB, et al. Evolving values in ethics and global health research. Glob Public Health. 2010;5(2):154–63. Consent for publication Not applicable. 23. Pratt B, Loff B. Linking International Research To Global Health Equity: The Limited Contribution Of Bioethics. Bioethics. 2013;27(4):208–14. 24. Kubanyiova M. Rethinking Research Ethics in Contemporary Applied Linguistics: The Tension Between Macroethical and Microethical Perspectives in Situated Research. Mod Lang J. 2008;92(4):503–18. Ethics approval and consent to participate Ethical approval was granted by the Royal College of Surgeons in Ireland Research Ethics Committee, and by the University of Zambia Humanities and Social Sciences Research Ethics Committee. Informed consent was given (through an information leaflet and signed consent form) by each interviewee. 25. Geissler PW, Molyneux C. Evidence, Ethos and Experiment. The Anthropology and History of Medical Research in Africa. 2011. 26. Emanuel EJ, et al. What makes clinical research in developing countries ethical? The benchmarks of ethical research. J Infect Dis. 2004;189(5):930–7 Availability of data and materials 17. Jentsch B, Pilley C. Research relationships between the South and the North: Cinderella and the ugly sisters? Soc Sci Med. 2003;57(10):1957–67. 17. Jentsch B, Pilley C. Research relationships between the South and the North: Cinderella and the ugly sisters? Soc Sci Med. 2003;57(10):1957–67. The transcripts generated for the study are not publicly available because consent was not obtained from participants for the release of the data. 18. Geissler PW. Public secrets in public health: Knowing not to know while making scientific knowledge. Am Ethnol. 2013;40(1):13–34. 18. Geissler PW. Public secrets in public health: Knowing not to know while making scientific knowledge. Am Ethnol. 2013;40(1):13–34. Received: 12 August 2016 Accepted: 28 November 2016 p 2. Research to involve at least two countries. Acknowledgements 14. Gonzalez Block MA, Mills A. Assessing capacity for health policy and systems research in low and middle income countries. Health Res Policy Systems. 2003;1(1):1. The authors would like to thank all the interviewees for taking time out to participate in the study. Sincere appreciation also goes to Chishimba Mulambia, the Zambian collaborator in the study. 14. Gonzalez Block MA, Mills A. Assessing capacity for health policy and systems research in low and middle income countries. Health Res Policy Systems. 2003;1(1):1. 15. Rottingen JA, et al. Mapping of available health research and development data: what's there, what's missing, and what role is there for a global observatory? Lancet. 2013;382(9900):1286–307. 15. Rottingen JA, et al. Mapping of available health research and development data: what's there, what's missing, and what role is there for a global observatory? Lancet. 2013;382(9900):1286–307. References Ethics in practice: the state of the debate on promoting the social value of global health research in resource poor settings particularly Africa. BMC Med Ethics. 2011;12:22. 4. KFPE, A Guide for Transboundary Research Partnership. 12 Principles S.C.f.R. P.w.D. Countries, Editor 2012. 4. KFPE, A Guide for Transboundary Research Partnership. 12 Principles S.C.f.R. P.w.D. Countries, Editor 2012. 4. KFPE, A Guide for Transboundary Research Partnership. 12 Principles S.C.f.R. P.w.D. Countries, Editor 2012. 5. Larkan F, et al. Developing a framework for successful research partnerships in global health. Glob Health. 2016;12(1):1–9. 5. Larkan F, et al. Developing a framework for successful research partnerships in global health. Glob Health. 2016;12(1):1–9. 2The literature review identified 13 evaluations of north– south health research, of which 9 were self-evaluations. 6. Provenzano AM, et al. Short-term global health research projects by US medical students: ethical challenges for partnerships. Am J Trop Med Hyg. 2010;83(2):211–4. 3The South African researcher was placed in the ‘north-south’ category. South African researchers are often considered to be northern by other African re- searchers, but considered to be southern by most north- ern researchers. 7. Hunt MR, Godard B. Beyond procedural ethics: Foregrounding questions of justice in global health research ethics training for students. Glob Public Health. 2013;8(6):713–24. 8. Kok MO, et al. The emergence and current performance of a health research system: lessons from Guinea Bissau. Health Res Policy Syst. 2012;10:5. 8. Kok MO, et al. The emergence and current performance of a health research system: lessons from Guinea Bissau. Health Res Policy Syst. 2012;10:5. 4It should be noted that other models of funding exist, where funding is channelled directly south, such as the Alliance for Health Policy and Systems Research. This model was not represented within this study sample. 9. Garrafa V, Lorenzo C. Moral imperialism and multi-centric clinical trials in peripheral countries. Cad Saude Publica. 2008;24(10):2219–26. 10. Adam T, et al. Trends in health policy and systems research over the past decade: still too little capacity in low-income countries. PLoS One. 2011; 6(11):e27263. 10. Adam T, et al. Trends in health policy and systems research over the past decade: still too little capacity in low-income countries. PLoS One. 2011; 6(11):e27263. 11. Molyneux S, Geissler PW. Ethics and the ethnography of medical research in Africa. Soc Sci Med. 2008;67(5):685–95. 11. Molyneux S, Geissler PW. Ethics and the ethnography of medical research in Africa. Soc Sci Med. 2008;67(5):685–95. 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https://openalex.org/W2012168161	https://rbho.emnuvens.com.br/rbho/article/download/706/513	Portuguese	null	Produção de mudas de Zínia elegans em substratos à base de resíduos agroindustriais e agropecuários em diferentes tamanhos de recipientes	Revista Brasileira de Horticultura Ornamental	2,011	cc-by	5,510	(1) Recebido em 24/07/2010 e aceito para publicação em 21/10/2010; (2) Parte da dissertação de mestrado do primeiro autor; (3) Aluno de Pós-Graduação em Solos e Nutrição de Plantas ESALQ/USP, Av. Pádua Dias 11, CEP 13418-900, Piracicaba (SP), hhfsousa@gmail.com; (4) Engº Agrônomo Dr.Sc Pesquisador da Embrapa Agroindústria Tropical, Rua Dra. Sara Mesquita, 2270, CEP 60.511-110, Fortaleza/CE, fred@cnpat. embrapa.br; Engº Agrônomo Dr. Sc Pesquisador da Embrapa Agroindústria Tropical, lindberg@cnpat.embrapa.br; (5) Engº Agrônomo Dr.Sc Professor do Departamento de Ciências do Solo da Universidade Federal do Ceará (UFC), Av. Mr. HUll, 2877, Campus do Pici, CEP 60.021-970, Fortaleza/CE, assisjr@ufc.br; (6) Aluno de Graduação em Agronomia da UFC. Production of Zínia elegans seedlings using organic by-products as substrate in different container size The substrate and container size are very important factors to obtain seedlings of good quality. The objectives of this study were to test eight substrates and three container sizes on the production of Zinia seedlings. The substrates were formulated by composing sugar cane bagace + bovine manure (compost 1), compost from fruits and vegetables waste + bovine manure (compost 2), green coir dust, wax palm straw and soil. The experiment was carried out under greenhouse conditions and the substrates tested were: S1= compost 1 + wax palm straw + soil (1:2:1), S2= compost 1 + green coir dust + soil (1:2:1), S3= compost 1 + wax palm straw (1:1), S4= compost 1 + green coir dust (1:1), S5= only compost 2, S6= compost 2 + wax palm straw (1:1), S7= compost 2 + green coir dust and S8= commercial substrate. The container tested were plastic trays with 228 cells (14 ml/cell), 150 cells (19 ml/cell) and 126 cells (30ml/cell) respectively. The percentage of seed germination was similar for all substrates and trays. The substrates S1 and S8 and tray with 126 cells (30 ml/cell) showed the best results for height, shoot dry matter and survival percentage of the seedlings. Keywords: floriculture, composting, recycling. RESUMO O substrato e o tamanho do recipiente são fatores importantes para a obtenção de mudas de qualidade. Os objetivos desse trabalho foram testar oito substratos e três tipos de recipientes na produção de mudas de zínia. Os substratos foram formulados com os seguintes compostos: bagaço de cana e esterco bovino (composto 1); lixo de frutas e de verduras mais esterco bovino (composto 2); e pó da casca de coco verde, bagana de carnaúba e solo. O experimento foi conduzido em condições de casa de vegetação, e os substratos testados foram: S1= composto 1 + bagana + solo (1:2:1); S2= composto 1 + pó de coco verde + solo (1:2:1); S3= composto 1 + bagana (1:1); S4= composto 1 + pó de coco verde (1:1); S5= composto 2 puro; S6= composto 2 + bagana (1:1); S7= composto 2 + pó de coco verde (1:1); e S8= substrato comercial. Os recipientes testados foram bandejas plásticas com 228 (14 ml/célula), 150 (19 ml/célula) e 126 células (30 ml/célula), respectivamente. A percentagem de germinação foi semelhante para todos os substratos e bandejas testados. Os substratos S1 e S8 e a bandeja com 126 células mostraram os melhores resultados para as variáveis altura, massa seca da parte aérea e percentagem de sobrevivência da mudas. l Palavras-chave: floricultura, compostagem, reciclagem. ABSTRACT Production of Zínia elegans seedlings using organic by-products as substrate in different container size 1. INTRODUÇÃO A reciclagem de materiais descartados é uma maneira de se evitar e/ou reduzir o efeito negativo destes resíduos, contribuindo com o desenvolvimento sustentável, agregando valores e diminuindo a utilização dos recursos naturais. São resíduos que podem servir de adubo e/ou substrato através do processo de compostagem. Os resíduos provenientes da Ceasa eram constituídos por materiais impróprios para consumo humano, como restos de verduras, frutas e legumes. O bagaço de cana foi proveniente da indústria de açúcar e álcool da região. No processo de compostagem, esses materiais foram triturados (entre 5 e 10 cm) e homogeneizados, em seguida, misturados com o esterco e colocado, em anéis de concreto protegido com telas para evitar a entrada de pequenos animais e insetos. Foi feito o monitoramento da temperatura e da umidade, sendo controladas com revolvimentos e irrigações sempre que necessário. O processo de compostagem teve duração de 60 dias, quando não mais foi observada variação na temperatura. Muitos materiais podem ser usados como substrato agrícola. Nesse sentido, estudos de ABAD et al. (2001) mostraram que de 105 materiais (resíduos) por eles avaliados, 63 apresentaram potencial para uso como substrato para espécies ornamentais. Substrato pode ser definido como o meio de cultivo de plantas em recipientes que podem ser constituídos por um ou vários materiais em misturas, inclusive resíduos diversos, devendo oferecer condições favoráveis para o bom crescimento das plantas. Após a compostagem, foram determinadas as condutividades elétricas (CEes) dos compostos 01 e 02, que apresentaram, respectivamente, 3,0 e 0,91 dS.m-1, e com base nesses valores foram determinadas as proporções na composição dos substratos testados. Para determinação da CEes, foi utilizada a solução extraída, após agitação, de uma mistura de substrato e água na relação de 1:5 (volume/ volume). No caso de produção de mudas, o volume dos recipientes exerce grande influência sobre o seu desenvolvimento, principalmente no que se refere à área de exploração pelas raízes, disponibilizando mais ou menos água e nutrientes para elas. A determinação do volume ideal dos recipientes para produção de mudas pode ser um grande diferencial no custo de produção, pois isso implica diretamente a quantidade de substrato que será utilizado. 1. INTRODUÇÃO Os substratos avaliados no trabalho foram formulados a partir dos produtos obtidos ao término do processo de compostagem, puros ou em misturas com outros materiais orgânicos ou solo: Substrato 01: Composto 01 + bagana de carnaúba + solo (1:2:1); Substrato 02: Composto 01 + pó de coco verde + solo (1:2:1); Substrato 03: Composto 01 + bagana de carnaúba (1:1); Substrato 04: Composto 01 + pó de coco verde (1:1); Substrato 05: Composto 02 puro; Substrato 06: Composto 02 + Bagana de carnaúba (1:1); Substrato 07: Composto 02 + pó de coco verde (1:1); e Substrato 08: Comercial (Hortimix). Segundo CUNHA et al. (2005), recipientes de maiores volumes oferecem melhores condições para o desenvolvimento das mudas, contudo, eles somente devem ser utilizados para espécies que apresentem desenvolvimento lento, necessitando permanecer no viveiro por um longo tempo, ou quando se desejam mudas bem desenvolvidas. Segundo BEZERRA (2003), recipientes de menor volume reduzem o crescimento e o vigor das mudas e ainda restringem o desenvolvimento de sistema radicular, e mudas produzidas em recipientes pequenos, normalmente, são menores e menos vigorosas do que aquelas produzidas em recipientes maiores. Após a formulação dos substratos, foram coletadas amostras para determinação dos atributos químicos, seguindo a metodologia usada pelo Laboratório de Solos e Água da Embrapa-CNPAT, que é baseada na Instrução Normativa Nº 46 do Ministério da Agricultura, Pecuária e Abastecimento. Todas as análises foram feitas em três repetições. A espécie Zinnia elegans Jacq. é uma planta herbácea, anual, de pleno sol, pertencente à família Asteraceae, popularmente conhecida como capitão, moça e velha ou canela-de-velho, apresenta flores do tipo margarida simples, dobrado ou crespo, sendo utilizada em parques e jardins de regiões tropicais e subtropicais. É uma ornamental cultivada também para a produção de flor de corte devido à sua longa durabilidade, sendo adequada também para a utilização em bordaduras e maciços a pleno sol em regiões de temperatura amena e áreas tropicais. Os recipientes testados foram bandejas de poliestireno laminado com 126 células (volume 30 ml/célula), R1; com 150 células (volume 19 ml/célula), R2; e com 228 células (volume 14 ml/célula), R3. A semeadura foi feita colocando-se três sementes de zínia por célula, deixando-se apenas uma planta após o raleio. Após a germinação, as plantas foram levadas para uma casa de vegetação sombreada a 50 % e mantidas neste ambiente durante toda a duração do experimento. 1. INTRODUÇÃO nordeste a triplicar em relação às aludidas regiões. No Nordeste brasileiro, vários resíduos rurais e urbanos com potencial para serem utilizados na agricultura já são usados por produtores agrícolas dessa região, porém a utilização desses materiais nem sempre apresenta bons resultados, pois suas características são desconhecidas e faltam estudos que possibilitem utilizá-los de forma racional para garantir a sustentabilidade econômica dos produtores em consonância com os princípios e objetivos do desenvolvimento sustentável. A floricultura ganhou um impulso significativo nos últimos anos, principalmente na região Nordeste, onde a produção e a comercialização vêm apresentando um incremento significativo na economia regional. Junto com esse aumento, surgiu a necessidade de estudos que viabilizassem essa atividade, tornando-a rentável e atrativa aos produtores. Por ser uma atividade intensiva com custos relativamente elevados, a utilização de mudas de qualidade é essencial para o sucesso na exploração de qualquer espécie ornamental. Nesse contexto, o substrato e o tamanho do recipiente onde são produzidas as mudas são muito importantes. A geração de resíduos, que é inerente à atividade do ser humano, constitui atualmente um dos grandes problemas enfrentados pelo homem, representando riscos para o ambiente e para a população, devido, principalmente, à contaminação das reservas hídricas e do solo, como também proporcionando um ambiente favorável para proliferação de organismos nocivos ao homem. Sua destinação é motivo de preocupação há algum tempo, porém, ela se agravou Os produtores e viveiristas do Nordeste enfrentam problemas com o alto custo de insumos, incluindo- se os substratos, cuja produção está concentrada nas regiões Sul e Sudeste do Brasil, chegando seu preço no V. 17, Nº.2, 2011, 115-120 V. 17, Nº.2, 2011 Revista Brasileira de Horticultura Ornamental Revista Brasileira de Horticultura Ornamental 116 Produção de mudas de Zinia elegans em substratos à base de resíduos agroindustriais e agropecuários em diferentes tamanhos de recipient Produção de mudas de Zínia elegans em substratos à base de resíduos agroindustriais e agropecuários em diferentes tamanhos de recipient com o surgimento dos grandes centros urbanos nas últimas décadas. Segundo MELO et al. (2000), a geração de lixo é um dos fatores que mais contribuem para a degradação do ambiente. foi conduzido de acordo com Kiehl (2002) em anéis de concreto, acomodados em ambiente coberto e pavimentado com cimento. Foram formulados dois compostos: Composto 01: Resíduos da Ceasa + esterco de gado (3:1) e Composto 02: Bagaço de cana + esterco de gado (3:1). 1. INTRODUÇÃO Os objetivos deste trabalho foram avaliar substratos formulados a partir de resíduos orgânicos de atividades agropecuárias e agroindustriais, compostados ou não, na produção de mudas de Zinia elegans, em diferentes tamanhos de recipientes. A irrigação foi realizada com um pulverizador manual, objetivando maior uniformidade na distribuição da água de acordo com a necessidade, até que a água começasse a escorrer pelo orifício da célula, sem, no entanto, ocorrer lixiviação (lavagem do substrato). Revista Brasileira de Horticultura Ornamental Revista Brasileira de Horticultura Ornamental HELON HÉBANO DE FREITAS SOUSA, FRED CARVALHO BEZERRA, RAIMUNDO NONATO DE ASSIS JÚNIOR et al HELON HÉBANO DE FREITAS SOUSA, FRED CARVALHO BEZERRA, RAIMUNDO NONATO DE ASSIS JÚNIOR et al. 117 Foi avaliada a percentagem de germinação no 7º dia após o semeio, e de acordo com informações na embalagem das sementes a germinação da espécie ocorria entre 5 – 10 dias após o semeio. E ao final do experimento (21 dias), foram avaliados a percentagem de sobrevivência, altura, número de folhas e produção de massa seca das mudas. A massa seca foi determinada após ter sido retirado o substrato aderido às raízes e em seguida seca em estufa a 65º C até massa constante. por um material mais fibroso e de difícil compostagem, o bagaço de cana-de-açúcar. Os substratos S1 e S8 apresentaram teores de nitrogênio, cálcio e magnésio maiores do que os demais substratos (Tabela 2). q ( ) O substrato que apresentou o segundo melhor resultado para as variáveis altura e produção de massa seca foi o S3, e para variável altura, ele foi igual ao S6; para a variável massa seca, o substrato S6 apresentou resultados inferiores ao substrato S3. O resíduo comum na composição dos substratos S1, S2, S3 e S6 é a bagana de carnaúba, subproduto da extração do pó da palha de carnaúba para produção de cera, utilizado, empiricamente, como cobertura morta na produção de hortaliças e recentemente na produção de plantas ornamentais, tendo esse resíduo favorecido o crescimento das mudas nos substratos que o continham. Esse efeito pode estar relacionado com a melhoria das propriedades físicas dos substratos, principalmente na macro e microporosidade, devido ao tamanho de suas partículas, ou ainda, ao aporte de nutrientes pela bagana. Pode-se observar relação entre a presença desse componente, independentemente dos compostos 01 e 02, e uma maior disponibilidade de alguns minerais como o nitrogênio, o fósforo e o potássio em relação àqueles formulados com os compostos 01 e 02 misturados ao pó de coco verde. TERCEIRO NETO (2004), estudando os teores de nutrientes totais, nutrientes solúveis em água e em Mehlich 1 em diferentes substratos na produção de mudas de violeta africana (Saintpaulia ionantha Wendl), observou que a bagana de carnaúba apresentou teores de nutrientes próximos àqueles observados nos substratos comerciais testados no mesmo trabalho, principalmente N, P e K. O delineamento experimental utilizado no trabalho foi o inteiramente casualizado com três tratamentos nas parcelas principais (tamanho de recipiente) e oito tratamentos nas subparcelas (substratos), em quatro repetições. Foram consideradas dez plantas como uma unidade experimental em esquema de parcelas subdivididas. 3. RESULTADOS E DISCUSSÃO A percentagem de germinação das sementes (Tabela 3) foi semelhante para todos os substratos e recipientes testados, com exceção do substrato S2, mostrando que os substratos apresentaram condições favoráveis ao processo germinativo, como disponibilidade adequada de água e ar. A germinação é influenciada, além dos fatores inerentes à semente (dormência), por condições ambientais como luz, temperatura, disponibilidade de água e oxigênio. A disponibilidade de água e oxigênio no substrato está relacionada com a textura, substratos com textura grosseira retêm menos água do que aqueles que apresentam granulometria mais fina; por outro lado, substratos que têm em sua composição partículas pequenas, implicando uma predominância de microporosidade, podem comprometer a disponibilidade de oxigênio necessário à germinação. O substrato adequado para germinação deve apresentar um equilíbrio entre a macro e a microporosidade, o que promove uma boa disponibilidade de água e oxigênio. De acordo com FERREIRA et al. (2008), um bom substrato deve proporcionar condições ideais para uma maior taxa de germinação e favorecer o crescimento das raízes. Para os demais substratos, os valores para essas variáveis foram estatisticamente inferiores aos substratos S1, S8, S3 e S6 e semelhantes entre si. Isso pode estar relacionado à baixa disponibilidade de nutrientes nos substratos S2 (composto 01 + pó de coco verde + solo), S4 (composto 01 + pó de coco verde), S5 (composto 02 puro) e S7 (composto 02 + pó de coco verde). De uma maneira geral, os substratos formulados com o composto 01 e bagana de carnaúba apresentaram os valores mais promissores, próximos ou iguais aos resultados obtidos com o substrato comercial para as variáveis testadas. LEAL et al. (2007) observaram melhores resultados na produção de mudas de alface, beterraba e tomate utilizando compostos orgânicos em comparação com um substrato comercial. Com relação à sobrevivência das mudas, observou-se, com exceção do S3, que os demais substratos se igualaram estatisticamente ao substrato comercial. Os resultados mostram que os maiores valores para as variáveis altura e produção de massa seca da parte aérea foram observados em mudas produzidas no substrato 01 (composto 01 + bagana + solo) e no substrato 08 (comercial), respectivamente, não havendo diferença estatística entre eles, seguido daqueles produzidos nos substratos 03 (composto 01 + bagana) e 06 (composto 02 + bagana). O número de folhas das mudas segue a mesma tendência do que foi observado nas variáveis altura e massa seca. HELON HÉBANO DE FREITAS SOUSA, FRED CARVALHO BEZERRA, RAIMUNDO NONATO DE ASSIS JÚNIOR et al HELON HÉBANO DE FREITAS SOUSA, FRED CARVALHO BEZERRA, RAIMUNDO NONATO DE ASSIS JÚNIOR et al. Cada bandeja contou com uma bordadura. Os resultados obtidos de todas as variáveis estudadas foram submetidos à análise de variância pelo teste F, sendo as médias comparadas pelo teste de Tukey a 5% de probabilidade. Revista Brasileira de Horticultura Ornamental Revista Brasileira de Horticultura Ornamental 2. MATERIAL E MÉTODOS O experimento foi conduzido em casa de vegetação situada nas dependências da Embrapa-CNPAT, sediada na cidade de Fortaleza, CE, Brasil. O experimento teve duração de 21 dias, tempo necessário para que as plântulas atingissem o tamanho para transplantio (5 cm). O processo de compostagem dos resíduos orgânicos V. 17, Nº.2, 2011, 115-120 V. 17, Nº.2, 2011 Revista Brasileira de Horticultura Ornamental Revista Brasileira de Horticultura Ornamental 3. RESULTADOS E DISCUSSÃO Observa-se que apenas as plantas produzidas nos substratos 01 e 08 apresentaram o segundo par de folhas definitivas, seguidas pelas plantas do substrato 03, diferentemente dos demais substratos que, em sua maioria, apresentaram apenas o primeiro par de folhas definitivas. Os resultados da germinação, sobrevivência, altura e massa seca foram iguais estatisticamente àqueles encontrados para as mudas produzidas no substrato comercial. O substrato S1 foi formulado com composto orgânico de resíduos da Ceasa, constituído por uma grande variedade de frutas e verduras frescas, o que pode ter conferido a esse composto uma melhor qualidade nutricional quando comparado ao composto 02, formado Com relação à caracterização dos substratos, os valores de pH (Tabela 1), em sua maioria, encontram-se fora das faixas consideradas ideais para meios de crescimento de acordo com VERDONCK et al. (1981), que sugerem uma faixa de 5,0 a 5,8; PENNINGSFELD (1978), de 5,5 a 6,5; VERDONCK (1983), de 4,0 a 6,5; e VERDONCK e GRABRIELS (1988), de 4,5 a 6,0. BENITO et al. (2006), trabalhando com substratos à base de compostados, V. 17, Nº.2, 2011, 115-120 V. 17, Nº.2, 2011 rodução de mudas de Zinia elegans em substratos à base de resíduos agroindustriais e agropecuários em diferentes tamanhos de recipientes rodução de mudas de Zínia elegans em substratos à base de resíduos agroindustriais e agropecuários em diferentes tamanhos de recipientes Produção de mudas de Zinia elegans em substratos à base de resíduos agroindustriais e agropecuários em diferentes tamanhos de recipient Produção de mudas de Zínia elegans em substratos à base de resíduos agroindustriais e agropecuários em diferentes tamanhos de recipient 118 encontraram valores superiores às faixas ótimas para meios de crescimento de plantas e sugeriram a mistura desses produtos a outros materiais que favoreçam a diminuição do pH desses substratos. com base no peso ou volume da mercadoria. O uso de recipientes menores funciona como uma alternativa a esse problema, diminuindo o peso e volume por muda, porém, segundo BEZERRA (2003), recipientes de menor volume reduzem o crescimento e o vigor das mudas e ainda restringem o desenvolvimento de sistema radicular. Mudas produzidas em recipientes pequenos, normalmente, são menores e menos vigorosas do que aquelas produzidas em recipientes grandes. O substrato 03 apresentou a maior condutividade elétrica, 1,88 dS m-1 (Tabela 1), observando-se influência da bagana no aumento desta variável. REFERÊNCIAS ABAD, M; NOGUERA, P.; BURÉS, S. National inventory of organic waste for use as growing media for ornamental potted plant production: case study in Spain. Bioresource Technology, n. 77, p. 197 – 200, 2001. ABAD, M; NOGUERA, P.; BURÉS, S. National inventory of organic waste for use as growing media for ornamental potted plant production: case study in Spain. Bioresource Technology, n. 77, p. 197 – 200, 2001. Resultados semelhantes aos encontrados nesse trabalho foram observados na aclimatização de mudas micropropagadas de abacaxi ornamental, em que foram usados diversos tamanhos de recipientes (BOMFIM, 2006). Segundo o autor, a superioridade dos recipientes de maior volume no desenvolvimento das plantas está diretamente relacionada às características intrínsecas dos recipientes como altura e capacidade volumétrica, traduzidas numa maior área para exploração pelas raízes, consequentemente, mais nutrientes, além da melhoria nas qualidades físicas como o espaço de aeração. BENITO, M.; MASAGUER, A.; MOLINER, A.; ANTONIO, R. de. Chemical and physical properties of pruning waste compost and their seazonal variability. Bioresource technology. n.97, p.2071-2076, 2006. , BEZERRA, F.C. Produção de mudas de hortaliças em ambiente protegido. Fortaleza: Embrapa Agroindústria Tropical, 2003. 22p. (Documentos, 72). BOMFIM, G.V. do. Efeito de lâminas e frequências de irrigação e tipos e volumes de substrato na aclimatação de mudas micropropagadas de abacaxizeiro ornamental. 167 f. il. color. enc. Dissertação (Mestrado em irrigação e drenagem) – Universidade Federal do Ceará, Fortaleza. 2006. TELLES et al. (2005), estudando diferentes volumes de substrato no desenvolvimento de Targetes patula, observaram que as plantas no maior volume de substrato apresentaram maior massa fresca e seca da parte aérea, sugerindo que o tamanho do recipiente influencia na disponibilidade de água e nutrientes e, consequentemente, no desenvolvimento da planta. CUNHA, A.O.; ANDRADE, L.A. de; BRUNO R. L. A.; SILVA, J. A. L. da; SOUZA, V.C. de; Efeitos de substratos e das dimensões dos recipientes na qualidade das mudas de Tabebuia impetiginosa (Mart. Ex D.C.) Standl. Revista Arvore, Viçosa, v.29, n.4, p.507-516, 2005. ´ Esses dados demonstram a importância da quantidade de substrato a ser explorado pelas raízes para produção de mudas de qualidade, pois recipientes maiores permitem um maior volume de raízes, aumentando a área de absorção de nutrientes. A utilização de uma adubação suplementar pode contornar esse problema de diminuição dos nutrientes devido à menor quantidade de substrato disponível, outros trabalhos mais específicos poderiam obter uma confirmação desse aspecto. FERREIRA, E.G.B. 3. RESULTADOS E DISCUSSÃO Comparando-se os valores das Ce do S1 e S2, nota-se que a presença da bagana foi responsável pelo ligeiro aumento da condutividade no substrato 01. Isso pode ser observado também quando comparados entre si os substratos 03 e 04, 06 e 07. Portanto, a escolha do tipo de recipiente a ser utilizado na formação de mudas deve ser baseada no custo de aquisição, na durabilidade do material, no tamanho e na forma, na facilidade de operação, na área ocupada dentro da área de cultivo e nas características para formação de mudas de boa qualidade (GONÇALVES, 1995; MACEDO, 1993). Os menores valores da CTC (Tabela 1) foram observados nos substratos 01 e 02, indicando que a presença de material mineral no substrato foi responsável por esses baixos valores. Diferentemente, os substratos formulados exclusivamente por material orgânico apresentaram valores de CTC elevados em virtude da grande superfície específica dos coloides orgânicos. O maior valor da CTC foi observado no composto comercial. Os resultados observados no trabalho sugerem que o recipiente R3 (126 células) e os substratos S1 (composto 1 + bagana + solo) e S8 (Hortimix) testados podem ser usados na produção de mudas de Zinnia elegans. O uso de resíduos na formulação de substratos implica diminuição dos custos de produção, reduzindo o impacto ambiental produzido pelo descarte desses materiais em lixões. Segundo os dados de sobrevivência, altura e produção de massa seca, observa-se que os diferentes tipos de recipiente influenciaram no desenvolvimento das mudas para essas variáveis, pois apresentam diferença significativa entre os tratamentos. Os resultados mais promissores foram aqueles observados nas mudas produzidas nas bandejas que continham células de 30 ml, seguidas daquelas produzidas em células de 19 e 14 ml para todos os substratos. Revista Brasileira de Horticultura Ornamental Revista Brasileira de Horticultura Ornamental REFERÊNCIAS de S.; MATOS, V.P.; SENA, L.H.de M.; SALES, A.G.F.A. Germinação de sementes e desenvolvimento de plântulas de crista de galo em diferentes substratos. Scientia Agraria, Curitiba, v.9, n.2, p.241-244, 2008. Atualmente existem diversas empresas especializadas na produção de mudas de hortaliças e plantas ornamentais. Essas empresas fornecem seus produtos para consumidores de diversas regiões do país, incluindo no preço final do produto o valor do transporte, que geralmente é calculado GONÇALVES, A.L. Substratos para produção de mudas de plantas ornamentais. In: MINAMI, K. (Ed). Produção GONÇALVES, A.L. Substratos para produção de mudas de plantas ornamentais. In: MINAMI, K. (Ed). Produção V. 17, Nº.2, 2011, 115-120 V. 17, Nº.2, 2011 HELON HÉBANO DE FREITAS SOUSA, FRED CARVALHO BEZERRA, RAIMUNDO NONATO DE ASSIS JÚNIOR et al HELON HÉBANO DE FREITAS SOUSA, FRED CARVALHO BEZERRA, RAIMUNDO NONATO DE ASSIS JÚNIOR et al. 119 g p y (p ) S1: composto 01 + bagana + solo (1:2:1); S2: composto 01 + pó de coco verde + solo (1:2:1); S3: composto 01 + bagana (1:1); S4: composto 01 + pó de coco verde; S5: composto 02 puro; S6: composto 02 + bagana (1:1); S7: composto 02 +pó de coco verde; S8: comercial. Valores seguidos de uma mesma letra nas colunas não diferem entre si pelo teste de Tuckey (p < 0,05) de mudas de qualidade em horticultura. São Paulo: T.A. Queiroz, 1995. 135p. TELLES, C.A.; MIELK, É. C.; MACHADO, M.P.; BIASE, L.A. Diferentes volumes de substrato no desenvolvimento de plantas de cravo de defunto (Targetes patula L). Revista Brasileira de Horticultura Ornamental, Campinas, v.11,n.1, p. 67-71, 2005. KIEHL, E. J. Conceitos sobre compostagem. In: Manual de Compostagem. Piracicaba: E.J. Kiehl, 3a edição do autor, 2002, p. 01 – 03. 171p. TERCEIRO NETO, C.P.C. Efeito da concentração da solução nutritiva e do substrato na aclimatação de plantas micropropagadas de violeta, 2004. 51 f. il. Dissertação (Mestrado em Solos e Nutrição de Plantas) – Universidade Federal do Ceará, Fortaleza. 2004. LEAL, M.A de A.; GUERRA, J.G.M.; PEIXOTO, R.T.G.; ALMEIDA, D.L. Utilização de compostos orgânicos como substrato na produção de mudas de hortaliças. Revista Horticultura Brasileira, v.25, n.3, p.392-395, 2007. MACEDO, A.L. Produção de mudas em viveiros florestais: espécies nativas. São Paulo: Fundação Florestal, 1993. 18 p. VERDONCK, O.; De VLEESCHAUWER, D,; De BOODT, M. The influence of the substrate to plant growth. Acta Horticulturae, Wageningen, n. 126, p.251-258, 1981. MELO, W.J. de, MARQUES, O.M.O.; MELO, V.P. de; CINTRA, A. A.D. Uso de resíduos em hortaliças e impacto ambiental. Revista Horticultura Brasileira, Brasília, v. 18, suplemento julho, p. 67 – 82, 2000. VERDONCK, O.; Reviewing and evaluation of new material used as substrates. Acta Horticulturae, Wageningen, n.150 , p.467-473, 1983. VERDONCK, O.; GABRIELS, R. Substrate requiriments for plants. Acta Horticulturae, Wageningen, n.221 , p.19- 23, 1988. abe a . p , C e C C dos subst atos testados Table 1. pH, EC and CEC of tested substrates Substrato pH Ce CTC dS.m-1 mmolc.L-1 SI 6,4 bc 1,27c 85,71g S2 7,3 abc 0,88d 109,75f S3 7,5ab 1,88a 165,22d S4 7,2abc 1,58b 242,22b S5 7,7a 0,91d 176,26cd S6 6,5bc 0,71e 143,80e S7 7,3abc 0,60e 191,29c S8 6,2c 0,98d 341,88a C.V. (%) 5,595 3,627 3,397 V. 17, Nº.2, 2011, 115-120 V. 17, Nº.2, 2011 Revista Brasileira de Horticultura Ornamental Revista Brasileira de Horticultura Ornamental Produção de mudas de Zinia elegans em substratos à base de resíduos agroindustriais e agropecuários em diferentes tamanhos de recipient Produção de mudas de Zínia elegans em substratos à base de resíduos agroindustriais e agropecuários em diferentes tamanhos de recipient 120 Tabela 2. Teores de nutrientes solúveis em água presentes nos substratos testados Table 2. Valores seguidas de uma mesma letra maiúscula nas linhas e minúsculas nas colunas não diferem entre si pelo teste de Tuckey (p < 0,05) S1: composto 01 + bagana + solo (1:2:1); S2: composto 01 + pó de coco verde + solo (1:2:1); S3: composto 01 + bagana (1:1); S4: composto 01 + pó de coco verde; S5: composto 02 puro; S6: composto 02 + bagana (1:1); S7: composto 02 +pó de coco verde; S8: comercial. R1: 228 células (14 ml); R2: 150 células (19 ml); R3: 126 células (30 ml). uidas de uma mesma letra maiúscula nas linhas e minúsculas nas colunas não diferem entre si pelo teste de Tuckey (p < 0,05) lores seguidos de uma mesma letra nas colunas não diferem entre si pelo teste de Tuckey (p < 0,05) g p y (p ) : composto 01 + bagana + solo (1:2:1); S2: composto 01 + pó de coco verde + solo (1:2:1); S3: composto 01 + bagana (1:1); S4: co coco verde; S5: composto 02 puro; S6: composto 02 + bagana (1:1); S7: composto 02 +pó de coco verde; S8: comercial. uidas de uma mesma letra maiúscula nas linhas e minúsculas nas colunas não diferem entre si pelo teste de Tuckey (p < 0,05) to 01 + bagana + solo (1:2:1); S2: composto 01 + pó de coco verde + solo (1:2:1); S3: composto 01 + bagana (1:1); S4: composto uma mesma letra maiúscula nas linhas e minúsculas nas colunas não diferem entre si pelo teste de Tuckey (p < 0,05) bagana + solo (1:2:1); S2: composto 01 + pó de coco verde + solo (1:2:1); S3: composto 01 + bagana (1:1); S4: composto 01 + pó de mudas de qualidade em horticultura. São Paulo: T.A. Queiroz, 1995. 135p. Water soluble nutrients content on tested substrates Substrato N-NH4+ N-NO3- P K Ca Mg Na S Cl g.L-1 SI 0,026 a 0,179 b 0,307 b 1,245 b 0,043 b 0,058 b 0,149 c 0,118 c 1,388 a S2 0,010 bc 0,003 e 0,340 a 0,854 c 0,018 d 0,031 e 0,149 c 0,103 cd 0,679 bc S3 0,011 b 0,136 c 0,267cd 2,583 a 0,034 c 0,056 b 0,224 b 0,231 b 1,477 a S4 0,003 d 0,119 d 0,205 e 1,120 b 0,025 d 0,024 f 0,268 a 0,221 b 1,388 a S5 0,006 cd 0,002 e 0,274 c 0,847 c 0,051 b 0,040 d 0,168 c 0,097 d 1,270 a S6 0,014 b 0,005 e 0,258 cd 1,185 b 0,026 cd 0,051 c 0,108 d 0,116 cd 0,679 bc S7 0,002 d 0,005 e 0,243 d 0,656 d 0,019 d 0,017 g 0,153 c 0,057 e 0,590 c S8 0,028 a 0,229 a 0,119 f 0,585 d 0,078 a 0,075 a 0,034 e 0,307 a 0,856 b C.V. (%) 13,58 6,32 3,337 4,378 7,945 4,294 6,819 4,808 8,862 Valores seguidos de uma mesma letra nas colunas não diferem entre si pelo teste de Tuckey (p < 0,05) S1: composto 01 + bagana + solo (1:2:1); S2: composto 01 + pó de coco verde + solo (1:2:1); S3: composto 01 + bagana (1:1); S4: composto 01 + pó de coco verde; S5: composto 02 puro; S6: composto 02 + bagana (1:1); S7: composto 02 +pó de coco verde; S8: comercial. Tabela 2. Teores de nutrientes solúveis em água presentes nos substratos testados Table 2. Water soluble nutrients content on tested substrates Valores seguidos de uma mesma letra nas colunas não diferem entre si pelo teste de Tuckey (p < 0,05) S1: composto 01 + bagana + solo (1:2:1); S2: composto 01 + pó de coco verde + solo (1:2:1); S3: composto 01 + bagana (1:1); S4: composto 01 + pó de coco verde; S5: composto 02 puro; S6: composto 02 + bagana (1:1); S7: composto 02 +pó de coco verde; S8: comercial. S1: composto 01 + bagana + solo (1:2:1); S2: composto 01 + pó de coco verde + solo (1:2:1); S3: composto 01 + bagana (1:1); S4: composto 01 + pó de coco verde; S5: composto 02 puro; S6: composto 02 + bagana (1:1); S7: composto 02 +pó de coco verde; S8: comercial. Tabela 3. de mudas de qualidade em horticultura. São Paulo: T.A. Queiroz, 1995. 135p. Percentagem de germinação de sementes, percentagem de sobrevivência, altura e massa seca de mudas de Zinia elegans em diferentes substratos e tamanhos de recipientes. Table 3. Seed germination percentage, survival percentage, height and dry matter of Zinia elegans seedlings in different substrates and container size Tabela 3. Percentagem de germinação de sementes, percentagem de sobrevivência, altura e massa seca de mudas de Zinia elegans em diferentes substratos e tamanhos de recipientes. Table 3. Seed germination percentage, survival percentage, height and dry matter of Zinia elegans seedlings in different substrates and container size. Tabela 3. Percentagem de germinação de sementes, percentagem de sobrevivência, altura e massa seca de mudas de Zinia elegans em diferentes substratos e tamanhos de recipientes. Table 3. Seed germination percentage, survival percentage, height and dry matter of Zinia elegans seedlings in different substrates and container size. Substrato S1 S2 S3 S4 S5 S6 S7 S8 Média Germinação (%) R1 100 95 100 100 100 100 100 100 99,37 a R2 100 100 100 100 100 100 100 100 100,00 a R3 100 100 100 100 100 100 100 100 100,00 a Média 100,00A 98,33B 100,00A 100,00A 100,00A 100,00A 100,00A 100,00A Sobrevivência (%) R1 97,5 80 75 95 95 87,5 97,5 90 89,69 b R2 100 100 67,5 97,5 97,5 90 95 95 92,81 ab R3 95 92,5 90 100 100 97,5 100 100 96,87 a Média 97,50 A 90,83 AB 77,50 B 97,50 A 97,50 A 91,67 A 97,50 A 95,00 A Altura (mm) R1 61,35 36,72 47,55 38,84 43,28 44,12 40,86 55.40 46,02 b R2 53,17 41,71 44,41 37,39 44,26 49,61 44,85 50,66 45,71 b R3 61,87 37,91 42,47 41,48 44,75 47,6 42,73 60,03 48,61 a Média 58,80 A 38,78 C 48,15 B 39,23 C 44,10 BC 47,11 B 42,69 BC 55,36 A Massa seca (g) R1 0,347 0,122 0,248 0,125 0,144 0,149 0,122 0,330 0,198c R2 0,428 0,149 0,325 0,130 0,143 0,191 0,153 0,395 0,239b R3 0,520 0,141 0,381 0,150 0,166 0,197 0,153 0,629 0,292a Média 0,431A 0,137C 0,318B 0,135C 0,151C 0,179C 0,143C 0,451A V l id d l iú l li h i ú l l ã dif i l d T k ( 0 05) Revista Brasileira de Horticultura Ornamental Revista Brasileira de Horticultura Ornamental V. 17, Nº.2, 2011, 115-120 V. 17, Nº.2, 2011
https://openalex.org/W2223235680	https://europepmc.org/articles/pmc4489761?pdf=render	English	null	Vitamin D Antagonises the Suppressive Effect of Inflammatory Cytokines on CTLA-4 Expression and Regulatory Function	PloS one	2,015	cc-by	10,465	Vitamin D Antagonises the Suppressive Effect of Inflammatory Cytokines on CTLA-4 Expression and Regulatory Function Louisa E. Jeffery1, Omar S. Qureshi1, David Gardner1, Tie Z. Hou2, Zoe Briggs1, Blagoje Soskic2, Jennifer Baker1, Karim Raza1,3‡, David M. Sansom2‡ 1 Medical Research Council Centre for Immune Regulation, School of Immunity and Infection, Institute of Biomedical Research, University of Birmingham Medical School, Birmingham, United Kingdom, 2 UCL Institute of Immunity and Transplantation, Royal Free Campus, University College London, London, United Kingdom, 3 Department of Rheumatology, Sandwell and West Birmingham Hospitals NHS Trust, Birmingham, United Kingdom a1111 ‡ These authors are joint senior authors on this work. k.raza@bham.ac.uk ‡ These authors are joint senior authors on this work. * k.raza@bham.ac.uk Abstract The immune suppressive protein CTLA-4 is constitutively expressed by Tregs and induced in effector T cells upon activation. Its crucial role in adaptive immunity is apparent from the fatal autoimmune pathology seen in CTLA-4 knockout mice. However, little is known regarding factors that regulate CTLA-4 expression and their effect upon its function to remove CD80 and CD86 from antigen presenting cells by transendocytosis. Th17 cells are emerging as significant players in autoimmunity as well as other diseases. Therefore, in this study we have examined the effects of Th17 polarising conditions on CTLA-4 expres- sion and function in human T cells and show that Th17 conditions can suppress the expression of CTLA-4 and its transendocytic function. In contrast to Th17 cells, vitamin D is inversely associated with autoimmune disease. We have previously shown a striking ability of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) to enhance CTLA-4, however, its effects upon B7 transendocytosis and its activity in the context of inflammation remained unknown. Here we show that induction of CTLA-4 by 1,25(OH)2D3 can actually be enhanced in the presence of Th17 polarising cytokines. Furthermore, its transendocytic function was maintained such that T cells generated in the presence of Th17 conditions and 1,25(OH)2D3 were highly effective at capturing CTLA-4 ligands from antigen present- ing cells and suppressing T cell division. Taken together, these data reveal an inhibitory effect of Th17 polarising conditions upon CTLA-4-mediated regulation and show that 1,25 (OH)2D3 counteracts this effect. Given the importance of CTLA-4-mediated suppression in the control of autoimmune diseases, our novel data highlight the importance of vitamin D in inflammatory settings. RESEARCH ARTICLE OPEN ACCESS Citation: Jeffery LE, Qureshi OS, Gardner D, Hou TZ, Briggs Z, Soskic B, et al. (2015) Vitamin D Antagonises the Suppressive Effect of Inflammatory Cytokines on CTLA-4 Expression and Regulatory Function. PLoS ONE 10(7): e0131539. doi:10.1371/ journal.pone.0131539 Citation: Jeffery LE, Qureshi OS, Gardner D, Hou TZ, Briggs Z, Soskic B, et al. (2015) Vitamin D Antagonises the Suppressive Effect of Inflammatory Cytokines on CTLA-4 Expression and Regulatory Function. PLoS ONE 10(7): e0131539. doi:10.1371/ journal.pone.0131539 Received: April 16, 2015 Accepted: June 3, 2015 Published: July 2, 2015 Copyright: © 2015 Jeffery 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 and its Supporting Information files. ‡ These authors are joint senior authors on this work. * k.raza@bham.ac.uk Introduction CTLA-4 is a critical suppressive protein that is expressed constitutively by regulatory T cells (Treg) and is induced on conventional T cells following activation [1–4]. It functions to restrain inappropriate activation of autoreactive T cells and to restore T cell homeostasis fol- lowing activation. This crucial regulatory role of CTLA-4 is evident from the lethal lympho- proliferative phenotype of CTLA-4 knockout mice [5, 6]. Recently, we and others observed CTLA-4 genetic variants that affected the level of CTLA-4 protein in autoimmunity and immunodeficiency, which indicates the importance of CTLA-4 expression in controlling human disease [7, 8]. Understanding how CTLA-4 expression is regulated therefore holds potential for therapeutic advances in conditions characterised by inappropriate or excessive T cell activation. Numerous studies have investigated the mechanisms by which CTLA-4 functions, leading to a range of proposed models [9–11]. An important feature of CTLA-4 biology is its internalisation and cycling back to the plasma membrane as well as its traffick- ing to lysosomes for degradation [12–14]. Consistent with this biology, we recently observed that CTLA-4 is able to remove its ligands, CD86 and CD80, from APCs and target them for degradation in a process termed transendocytsosis [15]. In this way, CTLA-4 reduces the availability of its shared ligands for CD28 co-stimulation in a quantitative manner that depends on the level of CTLA-4 expression. Surprisingly, despite the crucial role of CTLA-4 in immune regulation, relatively little is known about how its expression level and transendo- cytic activity are controlled, including the influence of environmental factors or the cytokine milieu. Th17 cells, which play an important role in the clearance of certain extracellular and intra- cellular pathogens [16, 17], are differentiated under inflammatory cytokine conditions and their dysregulation contributes to the pathology of a range of autoimmune diseases [18–20]. Their differentiation is intriguing since it is closely related to that of Treg through the common involvement of TGFβ [21, 22] with cytokines such as IL-1β, IL-6 and IL-23 promoting a Th17 outcome in humans [23, 24]. The relationship between inflammatory Th17 conditions and CTLA-4 expression is currently not well understood. In contrast, vitamin D is emerging as an important regulator of inflammatory responses. Indeed, low vitamin D status is associated with an increased risk of inflammatory diseases, including multiple sclerosis, type 1 diabetes, rheu- matoid arthritis and systemic lupus erythematosis (reviewed in [25–29]). Introduction Furthermore, vitamin D supplementation in mouse models of autoimmunity has suggested both prophylactic and therapeutic benefit [30–33]. We have shown that production of Th17-related cytokines is inhibited by vitamin D whilst regulatory markers, including CTLA-4, Foxp3 and IL-10, are increased [34] suggesting that vitamin D antagonises inflammatory outcomes and promotes regulation. In order to achieve a regulatory effect in an autoimmune setting, vitamin D would need to be effective within an inflammatory milieu. We therefore sought to determine the impact of such an environment on the expression and function of CTLA-4. We show that pro-Th17 cytokines substantially reduce CTLA-4 expression and function. However, even under Th17 polarizing conditions, vitamin D continues to drive upregulation of CTLA-4, generating T cells with CTLA-4-dependent regula- tory function. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Arthritis Research UK funded the project. Grant number DKAA RCAN14657. Funding was awarded to DS and KR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. PLOS ONE \| DOI:10.1371/journal.pone.0131539 July 2, 2015 1 / 18 Regulation of CTLA-4 by Vitamin D and Inflammatory Cytokines Materials and Methods This study was approved by the University of Birmingham Ethics Committee and given approval number ERN_14–0446. 2 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0131539 July 2, 2015 Regulation of CTLA-4 by Vitamin D and Inflammatory Cytokines Cell isolation and culture PBMCs were isolated by Ficoll gradient centrifugation from fresh leukocyte reduction system cones provided by the National Blood Service, Birmingham, UK. PBMCs were washed twice with PBS and twice with MACS buffer (0.5% BSA, 2 mM EDTA in PBS) and re-suspended at 1 x 108 cells/ml for magnetic separation. Conventional CD4+CD25- T cells and CD14+ mono- cytes were enriched by negative selection using cell separation reagents (StemCell Technolo- gies). Greater than 95% purity was obtained as assessed by flow cytometry. T cells were cultured in serum free medium (CellGenix) supplemented with 50U/ml Penicil- lin and Streptomycin (Gibco, Life Technologies). To assess the effect of cytokines and 1,25 (OH)2D3 upon their phenotype, T cells were stimulated with antiCD3CD28 Dynabeads (Life- Technologies) at a ratio of 1 bead: 4 T cells in the presence of recombinant cytokines and sup- plements as described in the figure legends. Supplements were added at the following concentrations: IL-1β (10ng/ml, Peprotech), IL-6 (20ng/ml, Immunotools), IL-23 (10ng/ml R and D Systems), TGFβ (1ng/ml, R and D Systems), 1,25(OH)2D3 (10nM, Sigma Alrich) and anti-human CTLA-4 (ticilimumab) (20μg/ml, a generous gift from Pfizer). The carrier for 1,25 (OH)2D3 was ethanol. It was diluted 1 in 1000 (v/v) into the culture. This concentration did not affect the measured outcomes. For all other reagents the vehicle was PBS. Monocyte derived dendritic cells (DCs) were cultured from monocytes in RPMI containing 10% FBS (Biosera), 50U/ml Penicillin and Streptomycin, 200μM glutamine (Life Technologies) (RPMI-FBS) and supplemented with GM-CSF (800U/ml, Peprotech) and IL-4 (500U/ml Peprotech). 1x106 cells were plated per well of a 24 well culture plate. At two to three days, cells were supplemented with fresh medium containing IL-4 and GM-CSF and cultured for a further six or seven days before use. DCs were CD11C+, CD14- and up-regulated CD86, CD80, CD40 and HLA-DR upon maturation with LPS (100ng/ml (Sigma Aldrich). All cells were cultured at 37°C, 95% humidity and 5% CO2. Cell labelling For some experiments, T cells were labelled with cell trace proliferation dyes, CFDA-SE or cell trace violet (Molecular probes, Life Technologies). Cells were washed two times with PBS and re-suspended in cell proliferation dye. CFDA-SE labelling was performed for 10 minutes at room temperature after which cells were washed three times in RPMI-FBS. For cell trace violet labelling T cells were labelled with cell trace violet for 20 minutes at 37°C. 10% FBS RPMI was added to quench the labelling. After incubation for 5 minutes at 37°C and centrifugation at room temperature, cells were washed a further two times with 10% FBS RPMI. PLOS ONE \| DOI:10.1371/journal.pone.0131539 July 2, 2015 CD86 acquisition assay (transendocytosis) A Chinese Hamster Ovarian (CHO) cell line expressing green fluorescent protein-tagged human CD86 (CD86-GFP) was generated as previously described [15] and cultured in DMEM (Life Technologies, supplemented with 10% FBS (Biosera), 50U/ml Penicillin and Streptomy- cin, 200μM glutamine (Life Technologies). Cells were passaged every 3 days. To enable CD86-GFP to be measured exclusively in T cells at the end of the assay, CHO-cells were labelled with 5mM Cell Trace Far Red DDAO-SE tracking dye (Molecular probes, Life Tech- nologies) immediately before use. T cells were prepared by stimulating them for four days with antiCD3/CD28 Dynabeads beads under Th0 or Th17 conditions in the presence or absence of 1,25(OH)2D3. Beads were removed using a magnet (Easysep StemCell Technologies) and cells washed with PBS and resuspended in serum free medium (CellGenix). A proportion of T cells were cultured with anti-CTLA-4 (40μg/ml) for 30 minutes before assembling the T cell- CHO-CD86GFP co cultures. 50,000 T cells and 150,000 CHO cells were cultured in a 96 well round bottom plate for 4 hours and 0.5μg/ml anti-CD3 (clone OKT3) added to promote CTLA-4 cycling. Control wells to set the background CD86-GFP transfer were prepared in which 5μg/ml anti-CD86 (clone BU63) was included. 30 minutes before analysis, anti-CTLA- 4-PE (BD Biosciences) was added to label cycling CTLA-4. Cells were analysed live by FACS. T cells were selected by gating according to forward-scatter/side scatter and single cells selected by pulse width. CHO-cells were excluded by selecting only far-red negative cells. Total CD86 acquisition was calculated by frequency of CD86-GFP+ cells x median fluorescence intensity of CD86-GFP+ cells. Non CTLA-4-mediated CD86-GFP acquisition, as determined from aCTLA-4 blocking antibody cultures, was then subtracted. Flow cytometry Dead cells were labelled with near-IR LIVE/DEAD fixable dead cell stain (Molecular Probes, Life Technologies) before fixation. For analysis of total CTLA-4, Foxp3 and CD25, cells were fixed, permeabilised and stained with ebioscience Foxp3 staining buffers according to the manufacturer’s instructions. For analysis of cytokine expression, cells were re-stimulated with PMA (50ng/ml), and ionomycin (1μM) for 5 hours, with Brefeldin A (10μg/ml) present dur- ing the last 4 hours (all from Sigma Aldrich). Cells were fixed with 3% paraformaldehyde in PBS for 12 minutes followed by a 5 minute wash with PBS under centrifugation. Cells were then permeabilised with 0.1% saponin (Acros Organics) prepared in PBS and stained with cytokine detection antibodies. Cells were acquired on a Dako Cyan flow cytometer (Dako Cytomation) and data analysed using FlowJo software (Tree Star). All antibodies were pur- chased from ebioscience or BD Biosciences and expression quantified relative to the appropri- ate isotype control. 3 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0131539 July 2, 2015 Regulation of CTLA-4 by Vitamin D and Inflammatory Cytokines Real-time PCR Total RNA was extracted using the TRIzol method (Life Technologies/Invitrogen). A total of 0.5μg was reverse transcribed with random hexamers using TaqMan reverse transcription reagents (Life Technologies/Applied Biosystems). Quantitative real-time PCR for VDR and 18SrRNA was then performed on an Applied Biosystems 7900 machine using assays on demand from Applied Biosystems (18S rRNA, 4319413E; VDR Hs00172113_m1). Amplifica- tion of cDNAs involved incubation at 50°C for 2 minutes and 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. VDR mRNA expression was then cal- culated relative to 18SrRNA using the delta Ct method. Dendritic Cell CD80/CD86 downregulation assays Bead stimulated T cells, polarized for six days under Th0 or Th17 conditions in the presence or absence of 1,25(OH)2D3, were cell trace violet labelled and a proportion treated with anti- CTLA-4 (40μg/ml) for 30 minutes before combining with DCs. T cells and DCs were co-cul- tured overnight in 96 well flat bottom plates at a ratio of 1DC:10 T cells in the presence of 0.5μg/ml anti-CD3 (OKT3). At 24 hours, cells were stained for CD86, CD80, CD11C and CD40 (BD Biosciences) and analysed by flow cytometry. DCs were selected by scatter and as violet trace negative. Suppression assays T cell suppressors were prepared by stimulating CD4+CD25- T cells with beads for six days. Beads were removed using a magnet (Easysep StemCell Technologies) and suppressors labelled with CFDA-SE (Molecular Probes, Life Technologies) as described above. A fraction of the suppressors were incubated with antiCTLA-4 (40μg/ml) for 30 minutes before the addition of 4 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0131539 July 2, 2015 Regulation of CTLA-4 by Vitamin D and Inflammatory Cytokines autologous DCs and allogenic, unstimulated violet cell trace labelled CD4+CD25- ‘responder’ T cells. Cells were combined at a ratio of 1DC: 40 responders: 8 suppressors and 0.5μg/ml antiCD3 (OKT3) added. To control for cell number, CFDA-SE labelled unstimulated CD4 +CD25- T cells were used in place of suppressors in control cultures. After 5 days, responder cell proliferation was monitored by flow cytometry. Single cells were selected according to pulse width and responder T cells identified as violet trace +ve, CFDA-SE-ve. Statistical analysis GraphPad Prism 5.0a software (GraphPad) was used for graphical summary and statistical analysis was performed using SPSS statistics version 22. Non-parametric Wilcoxon tests were used to test significance between two conditions when multiple treatments had not been used and n>5. To test interactions between 1,25(OH)2D3 and cytokine treatments repeated mea- sures two factor within subject analysis with Huynf-Feldt correction was performed. For mark- ers that did not show interaction the two factor analysis was re-run in the absence of interaction. Where interaction was detected single factor repeated measures analysis was per- formed to determine the effect of cytokine treatment under control and 1,25(OH)2D3 condi- tions separately. The Shapiro-Wilk normality test and inspection of normal Q-Q plots were used to confirm that the data could be tested with these parametric models. For data sets that did not pass the normality test (IL-17, IFNγ and IL-10) the data were log10 transformed, since by this transformation the residuals from the mean became normally distributed. PLOS ONE \| DOI:10.1371/journal.pone.0131539 July 2, 2015 Th17 polarising cytokines reduce CTLA-4 expression Cell trace-labelled CD4+CD25- T cells were stimulated for four days with antiCD3CD28 beads under no cytokine supplement (Th0), with TGFβ alone or with the pro-Th17 cocktail (TGFβ with IL-1β, IL-6 and IL-23) as indicated and expression of total CTLA-4 and Foxp3 assessed by flow cytometry. A) Representative FACS plots showing CTLA-4 against Foxp3 expression and cell division, indicated by cell-trace dilution. B) Summary of CTLA-4 expression for 12 donor donors. Bars indicate mean values and error bars show standard deviation. Significance was tested by repeated measures, single factor within subject analysis (* = P<0.05, *** = P<0.001). doi:10.1371/journal.pone.0131539.g001 Fig 1. Th17 polarising cytokines reduce CTLA-4 expression. Cell trace-labelled CD4+CD25- T cells were stimulated for four days with antiCD3CD28 beads under no cytokine supplement (Th0), with TGFβ alone or with the pro-Th17 cocktail (TGFβ with IL-1β, IL-6 and IL-23) as indicated and expression of total CTLA-4 and Foxp3 assessed by flow cytometry. A) Representative FACS plots showing CTLA-4 against Foxp3 expression and cell division, indicated by cell-trace dilution. B) Summary of CTLA-4 expression for 12 donor donors. Bars indicate mean values and error bars show standard deviation. Significance was tested by repeated measures, single factor within subject analysis (* = P<0.05, *** = P<0.001). doi:10 1371/journal pone 0131539 g001 Fig 1. Th17 polarising cytokines reduce CTLA-4 expression. Cell trace-labelled CD4+CD25- T cells were stimulated for four days with antiCD3CD28 beads under no cytokine supplement (Th0), with TGFβ alone or with the pro-Th17 cocktail (TGFβ with IL-1β, IL-6 and IL-23) as indicated and expression of total CTLA-4 and Foxp3 assessed by flow cytometry. A) Representative FACS plots showing CTLA-4 against Foxp3 expression and cell division, indicated by cell-trace dilution. B) Summary of CTLA-4 expression for 12 donor donors. Bars indicate mean values and error bars show standard deviation. Significance was tested by repeated measures, single factor within subject analysis (* = P<0.05, *** = P<0.001). d i 10 13 1/j l 0131 39 001 doi:10.1371/journal.pone.0131539.g001 Th17 polarising cytokines reduce CTLA-4 expression To assess the effect of Th17 polarisation on the expression of CTLA-4, we stimulated human CD4+CD25- T cells with anti-CD3/CD28 beads under Th0 conditions (no added cytokines) with TGFβ or with the Th17 polarising cytokines, TGFβ, IL-1β, IL-6 and IL-23, as shown (Fig 1a and 1b). Serum free medium was used to avoid background effects of TGFβ. Despite the absence of serum, the frequency of live cells at the end of culture was 75.4±8.3% and was not influenced by the cytokine treatment (ANOVA, P = 0.52). Whilst addition of TGFβ alone strongly induced Foxp3 it did not affect expression of CTLA-4. However, when TGFβ was combined with the pro-Th17 cytokines, IL-1β, IL-6 and IL-23, which did not affect CTLA-4 without TGFβ (S1 Fig), a significant decrease in CTLA-4 expression was observed (Fig 1). This was not the result of altered kinetics, since CTLA-4 expression was reduced across all divisions as defined by cell trace peaks (Fig 1a). To examine whether loss of CTLA-4 expression was a consequence of the Th17 transcrip- tional program or a more general phenomenon seen in all T cells exposed to Th17 polarising cytokines regardless of differentiation status, we measured CTLA-4 expression in relation to cytokine expression by flow cytometry. CTLA-4 was expressed by approximately 80% of T cells stimulated under either Th0 or Th17 conditions (Fig 2a) and by the majority of cells that expressed IL-17 (Fig 2b). Notably Th17 polarising conditions reduced CTLA-4 expression in CTLA-4+IL-17- as well as CTLA+IL-17+ T cells (Fig 2c), suggesting that the suppressive effect of Th17 polarising conditions upon CTLA-4 is not limited to cells undergoing the Th17 pro- gram of differentiation but is a general phenomenon. We explored this further by examining effects of Th17 polarising cytokines on CTLA-4 expression in T cells defined by the expression of other pro-inflammatory cytokines or regulatory-associated FoxP3. Th17 polarising cyto- kines reduced CTLA-4 in cells expressing IFNγ, IL-21, TNFα and IL-2 (Fig 2d) as well as in FoxP3+ and FoxP3- T cells (Fig 2e). Thus pro-Th17 cytokines suppress CTLA-4 in T cells of different lineages, both regulatory and inflammatory. 5 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0131539 July 2, 2015 Regulation of CTLA-4 by Vitamin D and Inflammatory Cytokines Fig 1. Th17 polarising cytokines reduce CTLA-4 expression. 1,25(OH)2D3 promotes a Treg phenotype and increases CTLA-4 expression even under inflammatory conditions Given that we have previously demonstrated that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) can increase CTLA-4 expression, we next sought to determine if the inhibitory effect of Th17 polarising cytokines on CTLA-4 expression was maintained in the presence of 1,25(OH)2D3. 6 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0131539 July 2, 2015 Fig 2. Suppression of CTLA-4 by Th17 polarising cytokines is not specific to IL-17+ T cells. CD4 +CD25- T cells were stimulated in the presence of Th17 polarising cytokines for four days and assessed for IL-17, IFNγ, IL-21, TNFα, IL-2 or Foxp3 in combination with CTLA-4 by flow cytometry. A) Frequency of total CTLA-4+ cells. B) Representative bivariate FACS plot of CTLA-4 versus IL-17 for cells cultured under Th17 polarising conditions. C) CTLA-4 expression in CTLA-4+ T cells gated according to IL-17 expression. D) CTLA-4 expression by CTLA-4+ T cells that expressed IL-17, IFNγ, IL-21, TNFα or IL-2. E) CTLA-4 expression in CTLA-4+ T cells defined by FoxP3 expression. In C, D and E expression under Th17 conditions is expressed relative to the level under Th0 conditions. Data are summarised for n7 donors. Bars indicate median values and error bars show the semi interquartile range. Significance with respect to cells expressing the marker under Th0 conditions was tested by Wilcoxon matched paired tests. (* = P<0.05, = P<0.01, * = P<0.001). doi:10.1371/journal.pone.0131539.g002 Regulation of CTLA-4 by Vitamin D and Inflammatory Cytokines Regulation of CTLA-4 by Vitamin D and Inflammatory Cytokines Fig 2. Suppression of CTLA-4 by Th17 polarising cytokines is not specific to IL-17+ T cells. CD4 +CD25- T cells were stimulated in the presence of Th17 polarising cytokines for four days and assessed for IL-17, IFNγ, IL-21, TNFα, IL-2 or Foxp3 in combination with CTLA-4 by flow cytometry. A) Frequency of total CTLA-4+ cells. B) Representative bivariate FACS plot of CTLA-4 versus IL-17 for cells cultured under Th17 polarising conditions. C) CTLA-4 expression in CTLA-4+ T cells gated according to IL-17 expression. D) CTLA-4 expression by CTLA-4+ T cells that expressed IL-17, IFNγ, IL-21, TNFα or IL-2. E) CTLA-4 expression in CTLA-4+ T cells defined by FoxP3 expression. In C, D and E expression under Th17 conditions is expressed relative to the level under Th0 conditions. Data are summarised for n7 donors. Bars indicate median values and error bars show the semi interquartile range. 1,25(OH)2D3 promotes a Treg phenotype and increases CTLA-4 expression even under inflammatory conditions Significance with respect to cells expressing the marker under Th0 conditions was tested by Wilcoxon matched paired tests. (* = P<0.05, = P<0.01, * = P<0.001). CD4+CD25- T cells were therefore stimulated as before under Th0, TGFβ alone and Th17 conditions, either in the presence or absence of 10nM 1,25(OH)2D3. Notably, 1,25(OH)2D3 did not influence the frequency of live cells at the end of culture under any cytokine treat- ment (PD3>0.05 and PCyt>0.05). However, across cytokine treatments, 1,25(OH)2D3 pro- moted marked up-regulation of CTLA-4 expression (Fig 3a and S1 Table). Furthermore a CD4+CD25- T cells were therefore stimulated as before under Th0, TGFβ alone and Th17 conditions, either in the presence or absence of 10nM 1,25(OH)2D3. Notably, 1,25(OH)2D3 did not influence the frequency of live cells at the end of culture under any cytokine treat- ment (PD3>0.05 and PCyt>0.05). However, across cytokine treatments, 1,25(OH)2D3 pro- moted marked up-regulation of CTLA-4 expression (Fig 3a and S1 Table). Furthermore a 7 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0131539 July 2, 2015 Regulation of CTLA-4 by Vitamin D and Inflammatory Cytokines Fig 3. 1,25(OH)2D3 maintains a regulatory T cell phenotype even under inflammatory, Th17 polarising conditions. CD4+CD25- T cells were stimulated in the presence of recombinant cytokines IL-1β, IL-6, IL-23 and TGFβ as indicated with or without 1,25(OH)2D3 and expression of regulatory-associated markers CTLA- 4, Foxp3 and CD25 assessed at four days and cytokines IL-2, IL-17, IFNγ, IL-21 and IL-10 measured at five days by flow cytometry. Data are summarised for n5 donors. Bars indicate mean values and error bars show the standard deviation. Repeated measures, two factor within subject analysis was used to test interaction between 1,25(OH)2D3 and cytokine treatment (S1 Table). For markers that did not show interaction the two factor analysis was re-run in the absence of interaction and P values for each factor are shown (1,25(OH)2D3 = PD3 and cytokine treatment = PCyt. Where interaction was detected, single factor analysis was performed. P values are shown for the effect of cytokine treatment under control (Pcyt—D3) and 1,25(OH)2D3 (Pcyt + D3) conditions separately. Significant contrasts between cytokine treatments are indicated by stars (* = P<0.05, = P<0.01, * = P<0.001). Fig 3. 1,25(OH)2D3 maintains a regulatory T cell phenotype even under inflammatory, Th17 polarising conditions. 1,25(OH)2D3 promotes a Treg phenotype and increases CTLA-4 expression even under inflammatory conditions For IL-10, which we and others have previously shown to be induced by 1,25(OH)2D3, we detected a significant effect of 1,25 (OH)2D3 across cytokine treatments as well as a strong interaction. Single factor analysis also indicated that TGFβ and Th17 conditions strongly suppress IL-10 in the absence of 1,25 (OH)2D3, with Th17 conditions promoting strongest suppression. Under Th0, 1,25(OH)2D3 supressed IL-10. However, in the presence of 1,25(OH)2D3 both TGFβ and Th17 conditions increased IL-10. Thus, taken together, these data demonstrate that 1,25(OH)2D3 promotes a regulatory phenotype, with high CTLA-4, and CD25 and lack of inflammatory cytokines such as IL-17, IFNγ and IL-21. In the presence of TGFβ, FoxP3 was also enhanced and 1,25 (OH)2D3 raised IL-10. Most importantly, our data also show that even when inflammatory cytokines are present, 1,25(OH)2D3 has the dominant effect and a regulatory T cell pheno- type is maintained. Vitamin D receptor expression is increased by TGFβ and maintained in the presence of Th17 polarising cytokines Since 1,25(OH)2D3 exerts its effects through the steroidal nuclear vitamin D receptor (VDR), we investigated the impact of the above treatments on VDR expression. As shown in Fig 4, TGFβ increased VDR mRNA relative to control. This effect was maintained under Th17 condi- tions but inflammatory cytokines, IL-1β, IL-6 and IL-23, did not enhance VDR without TGFβ. Together these data support the hypothesis that increased VDR expression, seen under Th17 conditions, is the result of the influence of TGFβ. Moreover this suggests that the enhanced effect of 1,25(OH)2D3 on CTLA-4 expression under Th17 conditions, compared with Th0 con- ditions, may involve increased VDR expression. 1,25(OH)2D3 promotes a Treg phenotype and increases CTLA-4 expression even under inflammatory conditions CD4+CD25- T cells were stimulated in the presence of recombinant cytokines IL-1β, IL-6, IL-23 and TGFβ as indicated with or without 1,25(OH)2D3 and expression of regulatory-associated markers CTLA- 4, Foxp3 and CD25 assessed at four days and cytokines IL-2, IL-17, IFNγ, IL-21 and IL-10 measured at five days by flow cytometry. Data are summarised for n5 donors. Bars indicate mean values and error bars show the standard deviation. Repeated measures, two factor within subject analysis was used to test interaction between 1,25(OH)2D3 and cytokine treatment (S1 Table). For markers that did not show interaction the two factor analysis was re-run in the absence of interaction and P values for each factor are shown (1,25(OH)2D3 = PD3 and cytokine treatment = PCyt. Where interaction was detected, single factor analysis was performed. P values are shown for the effect of cytokine treatment under control (Pcyt—D3) and 1,25(OH)2D3 (Pcyt + D3) conditions separately. Significant contrasts between cytokine treatments are indicated by stars (* = P<0.05, = P<0.01, * = P<0.001). Fig 3. 1,25(OH)2D3 maintains a regulatory T cell phenotype even under inflammatory, Th17 polarising doi:10.1371/journal.pone.0131539.g003 doi:10.1371/journal.pone.0131539.g003 strong interaction between cytokine treatment and 1,25(OH)2D3 was observed indicating that the cytokine effect is different when 1,25(OH)2D3 is present. Subsequent inspection using single factor within subject analysis showed that whilst Th17 conditions suppressed CTLA-4 in the absence of 1,25(OH)2D3, when 1,25(OH)2D3 was present TGFβ and to a strong interaction between cytokine treatment and 1,25(OH)2D3 was observed indicating that the cytokine effect is different when 1,25(OH)2D3 is present. Subsequent inspection using single factor within subject analysis showed that whilst Th17 conditions suppressed CTLA-4 in the absence of 1,25(OH)2D3, when 1,25(OH)2D3 was present TGFβ and to a 8 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0131539 July 2, 2015 Regulation of CTLA-4 by Vitamin D and Inflammatory Cytokines greater extent Th17 conditions, enhanced CTLA-4 above the level induced by 1,25(OH)2D3 alone (Fig 3a and S2 Table). In addition to CTLA-4, we analysed the effect of 1,25(OH)2D3 under cytokine treatments upon other markers known to be sensitive to 1,25(OH)2D3, including FoxP3, CD25, IL-2, IL-17, IFNγ, IL-21, and IL-10 (Fig 3b–3h and S1 and S2 Tables). 1,25(OH)2D3 did not significantly affect the overall expression of FoxP3 or IL-2 but it did alter the magnitude of the cytokine effects (P interaction <0.05). Nonetheless, in both the presence and absence of 1,25(OH)2D3 inflammatory cytokines reduced the TGFβ driven increase of FoxP3 and IL-2. 1,25(OH)2D3 promotes a Treg phenotype and increases CTLA-4 expression even under inflammatory conditions For CD25, 1,25(OH)2D3 significantly increased expression across treatments but no interaction was detected. By contrast, across all cytokine backgrounds, 1,25(OH)2D3 significantly inhibited IL-17, IFNγ and IL-21 and for IL-17 an interaction between 1,25(OH)2D3 and cytokine treatment was detected, as 1,25(OH)2D3 reduced the extent of IL-17 up-regulation by TGFβ and Th17 treatments. For IL-10, which we and others have previously shown to be induced by 1,25(OH)2D3, we detected a significant effect of 1,25 (OH)2D3 across cytokine treatments as well as a strong interaction. Single factor analysis also indicated that TGFβ and Th17 conditions strongly suppress IL-10 in the absence of 1,25 (OH)2D3, with Th17 conditions promoting strongest suppression. Under Th0, 1,25(OH)2D3 supressed IL-10. However, in the presence of 1,25(OH)2D3 both TGFβ and Th17 conditions increased IL-10. Thus, taken together, these data demonstrate that 1,25(OH)2D3 promotes a regulatory phenotype, with high CTLA-4, and CD25 and lack of inflammatory cytokines such as IL-17, IFNγ and IL-21. In the presence of TGFβ, FoxP3 was also enhanced and 1,25 (OH)2D3 raised IL-10. Most importantly, our data also show that even when inflammatory cytokines are present, 1,25(OH)2D3 has the dominant effect and a regulatory T cell pheno- type is maintained. greater extent Th17 conditions, enhanced CTLA-4 above the level induced by 1,25(OH)2D3 alone (Fig 3a and S2 Table). In addition to CTLA-4, we analysed the effect of 1,25(OH)2D3 under cytokine treatments upon other markers known to be sensitive to 1,25(OH)2D3, greater extent Th17 conditions, enhanced CTLA-4 above the level induced by 1,25(OH)2D3 alone (Fig 3a and S2 Table). In addition to CTLA-4, we analysed the effect of 1,25(OH)2D3 under cytokine treatments upon other markers known to be sensitive to 1,25(OH)2D3, including FoxP3, CD25, IL-2, IL-17, IFNγ, IL-21, and IL-10 (Fig 3b–3h and S1 and S2 Tables). 1,25(OH)2D3 did not significantly affect the overall expression of FoxP3 or IL-2 but it did alter the magnitude of the cytokine effects (P interaction <0.05). Nonetheless, in both the presence and absence of 1,25(OH)2D3 inflammatory cytokines reduced the TGFβ driven increase of FoxP3 and IL-2. For CD25, 1,25(OH)2D3 significantly increased expression across treatments but no interaction was detected. By contrast, across all cytokine backgrounds, 1,25(OH)2D3 significantly inhibited IL-17, IFNγ and IL-21 and for IL-17 an interaction between 1,25(OH)2D3 and cytokine treatment was detected, as 1,25(OH)2D3 reduced the extent of IL-17 up-regulation by TGFβ and Th17 treatments. PLOS ONE \| DOI:10.1371/journal.pone.0131539 July 2, 2015 1,25(OH)2D3 promotes the suppressive function of CTLA-4 under Th17 conditions Data are from four donors. Bars show median values and error bars indicate semi interquartile range. doi:10.1371/journal.pone.0131539.g004 data reveal that whilst pro-inflammatory Th17 conditions reduce CTLA-4 expression and therefore transendocytic function, 1,25(OH)2D3 overrides this effect on both expression level and on transendocytosis, driving a T cell phenotype that has high capacity to remove CTLA-4 co-stimulatory ligands from target cells. data reveal that whilst pro-inflammatory Th17 conditions reduce CTLA-4 expression and therefore transendocytic function, 1,25(OH)2D3 overrides this effect on both expression level and on transendocytosis, driving a T cell phenotype that has high capacity to remove CTLA-4 co-stimulatory ligands from target cells. 1,25(OH)2D3 promotes the suppressive function of CTLA-4 under Th17 conditions Transendoytosis is the major mechanism via which CTLA-4 mediates T cell suppression. Thus, having observed that Th17 cytokines suppress CTLA-4, whilst 1,25(OH)2D3 strongly enhances it, we wished to determine how these treatments affected CTLA-4 trafficking and transendocytosis. For this, we used an in-vitro system in which Chinese Hamster Ovary (CHO) cells, stably expressing GFP-tagged CD86, were dye-labelled and co-cultured with CTLA-4 expressing T cell blasts. Anti-CD3 was included to promote T cell activation and stimulate transendocytosis (Fig 5a). Bivariate flow cytometry analysis of CTLA-4 versus CD86 capture clearly revealed that capture of CD86 by T cells was related to the level of CTLA-4 trafficking (Fig 5b). Accordingly, the percentage of T cells that acquired CD86 was reduced under Th17 conditions (P = 0.016, Wilcoxon test n = 7). In contrast, ligand capture was substantially enhanced in the presence of 1,25(OH)2D3 even under Th17 conditions (Fig 5c). Overall, these PLOS ONE \| DOI:10.1371/journal.pone.0131539 July 2, 2015 9 / 18 Regulation of CTLA-4 by Vitamin D and Inflammatory Cytokines Fig 4. Vitamin D receptor (VDR) expression is increased by TGFβ in the presence of Th17 polarising cytokines. CD4+CD25- T cells were stimulated with antiCD3CD28 antibodies for 18 hours in the presence of recombinant cytokines and 1,25(OH)2D3 as shown and VDR mRNA measured by qPCR. Expression was normalized to 18S RNA and is plotted relative to expression in the absence of recombinant cytokines or 1,25 (OH)2D3. Data are from four donors. Bars show median values and error bars indicate semi interquartile range. doi:10.1371/journal.pone.0131539.g004 Fig 4. Vitamin D receptor (VDR) expression is increased by TGFβ in the presence of Th17 polarising cytokines. CD4+CD25- T cells were stimulated with antiCD3CD28 antibodies for 18 hours in the presence of recombinant cytokines and 1,25(OH)2D3 as shown and VDR mRNA measured by qPCR. Expression was normalized to 18S RNA and is plotted relative to expression in the absence of recombinant cytokines or 1,25 (OH)2D3. Data are from four donors. Bars show median values and error bars indicate semi interquartile range. Fig 4. Vitamin D receptor (VDR) expression is increased by TGFβ in the presence of Th17 polarising cytokines. CD4+CD25- T cells were stimulated with antiCD3CD28 antibodies for 18 hours in the presence of recombinant cytokines and 1,25(OH)2D3 as shown and VDR mRNA measured by qPCR. Expression was normalized to 18S RNA and is plotted relative to expression in the absence of recombinant cytokines or 1,25 (OH)2D3. PLOS ONE \| DOI:10.1371/journal.pone.0131539 July 2, 2015 1,25(OH)2D3 enhances CTLA-4-mediated suppression in dendritic cell- driven stimulations Although CTLA-4-dependent ligand capture by T cells is an important indicator of suppressive capability, it does not necessarily reflect the level of ligand downregulation on the APC. Rather, effective ligand downregulation and therefore suppression ultimately integrates a number of parameters besides CTLA-4 expression, for example ligand synthesis rates, which could be influenced by cytokines such as IFNγ and IL-10 [35–37], both of which were modified by 1,25 (OH)2D3 (Fig 3c). With this in mind, we sought to test the significance of altered CTLA-4 lev- els, as controlled by inflammatory cytokines and 1,25(OH)2D3, upon ligand expression by co- cultured dendritic cells. T cells were stimulated under Th0 and Th17 conditions in the presence or absence of 1,25(OH)2D3, and then cultured overnight with DCs. DC expression of CD80, CD86, and the control markers CD11c and CD40 was then assessed by flow cytometry. As shown in Fig 6a, all activated T cells caused some downregulation of CD86 and CD80 but not control proteins. However, the magnitude of CTLA-4-dependent depletion was greatest for T cells cultured in the presence of 1,25(OH)2D3. Notably, depletion of CD80 was greater than that of CD86 in line with the higher affinity of CTLA-4 for CD80 [38]. Finally, we tested the ability of T cells cultured under Th17 and 1,25(OH)2D3 conditions to act as suppressor cells and affect the proliferation of responder T cells stimulated by DCs. Here we observed that Th0 and Th17 T cells had little effect upon the proliferation of responder T cells, with Th17 PLOS ONE \| DOI:10.1371/journal.pone.0131539 July 2, 2015 10 / 18 Regulation of CTLA-4 by Vitamin D and Inflammatory Cytokines Fig 5. Transendocytic function of CTLA-4 is promoted by 1,25(OH)2D3 and maintained under inflammatory conditions. CTLA-4 transendocytic function was tested as described in the methods. A) Gating strategy to ensure exclusion of CD86-GFP donor cells from the measurement of T cell GFP acquisition. B) Representative FACS plots of CD86-GFP acquisition versus trafficking CTLA-4. C) Total CTLA-4 mediated CD86 acquisition by T cells summarized for n = 5 donors. Bars indicate mean values and error bars show the standard deviation. P values for the separate effects of Th17 cytokines (Pcyt) and 1,25 (OH)2D3 (PD3) are shown as determined by the 2 factor without interaction model since no interaction was detected (P = 0.146). doi:10.1371/journal.pone.0131539.g005 Fig 5. Transendocytic function of CTLA-4 is promoted by 1,25(OH)2D3 and maintained under inflammatory conditions. 1,25(OH)2D3 enhances CTLA-4-mediated suppression in dendritic cell- driven stimulations 1,25(OH)2D3 promotes CTLA-4-mediated B7 depletion from dendritic cells and suppression of T cell proliferation. CTLA-4 expressing ‘suppressor cells’ were prepared by stimulating CD4+CD25- T cells under Th0 or Th17 conditions in the presence or absence of 1,25(OH)2D3. A) Suppressor cells were cultured with autologous DCs and antiCD3 for 24 hours with or without CTLA-4 blocking antibody. Expression of CD80, CD86, CD11c and CD40 by DCs was measured by flow cytometry. Dot plots show the ratio of marker expression in control versus anti-CTLA-4-treated cultures for four donors. B) Suppressor cells were CFDA-SE labeled and added to autologous DC plus antiCD3 stimulations of allogeneic cell trace violet labeled CD4+CD25- T cells (responders) with or without anti-CTLA-4. Parallel stimulations were also prepared in which CFDA-SE-labeled CD4+CD25- were added in place of suppressor T cells as a control for cell number. At five days, proliferation of responder T cells was assessed by flow cytometry. Data are from one donor but representative of four. Shaded histograms show proliferation in the absence of suppressors. Dotted and solid lines indicate proliferation in the presence versus the absence of anti-CTLA-4 respectively. Fig 6. 1,25(OH)2D3 promotes CTLA-4-mediated B7 depletion from dendritic cells and suppression of T cell proliferation. CTLA-4 expressing ‘suppressor cells’ were prepared by stimulating CD4+CD25- T cells under Th0 or Th17 conditions in the presence or absence of 1,25(OH)2D3. A) Suppressor cells were cultured with autologous DCs and antiCD3 for 24 hours with or without CTLA-4 blocking antibody. Expression of CD80, CD86, CD11c and CD40 by DCs was measured by flow cytometry. Dot plots show the ratio of marker expression in control versus anti-CTLA-4-treated cultures for four donors. B) Suppressor cells were CFDA-SE labeled and added to autologous DC plus antiCD3 stimulations of allogeneic cell trace violet labeled CD4+CD25- T cells (responders) with or without anti-CTLA-4. Parallel stimulations were also prepared in which CFDA-SE-labeled CD4+CD25- were added in place of suppressor T cells as a control for cell number. At five days, proliferation of responder T cells was assessed by flow cytometry. Data are from one donor but representative of four. Shaded histograms show proliferation in the absence of suppressors. Dotted and solid lines indicate proliferation in the presence versus the absence of anti-CTLA-4 respectively. doi:10.1371/journal.pone.0131539.g006 1,25(OH)2D3 enhances CTLA-4-mediated suppression in dendritic cell- driven stimulations CTLA-4 transendocytic function was tested as described in the methods. A) Gating strategy to ensure exclusion of CD86-GFP donor cells from the measurement of T cell GFP acquisition. B) Representative FACS plots of CD86-GFP acquisition versus trafficking CTLA-4. C) Total CTLA-4 mediated CD86 acquisition by T cells summarized for n = 5 donors. Bars indicate mean values and error bars show the standard deviation. P values for the separate effects of Th17 cytokines (Pcyt) and 1,25 (OH)2D3 (PD3) are shown as determined by the 2 factor without interaction model since no interaction was detected (P = 0.146). doi:10.1371/journal.pone.0131539.g005 doi:10.1371/journal.pone.0131539.g005 conditioned cells showing the least CTLA-4 mediated control. By marked contrast, T cells stimulated in the presence of 1,25(OH)2D3 under Th17 conditions as well as under Th0 condi- tions caused robust CTLA-4-mediated suppression (Fig 6b). Taken together, these data sup- port the view that CTLA-4 on activated T cells plays a role in regulating co-stimulation and that 1,25(OH)2D3 enhances this pathway. Furthermore, they confirm that the effect of 1,25 (OH)2D3 upon this pathway is maintained, even under inflammatory conditions. PLOS ONE \| DOI:10.1371/journal.pone.0131539 July 2, 2015 11 / 18 Regulation of CTLA-4 by Vitamin D and Inflammatory Cytokines Fig 6. 1,25(OH)2D3 promotes CTLA-4-mediated B7 depletion from dendritic cells and suppression of T cell proliferation. CTLA-4 expressing ‘suppressor cells’ were prepared by stimulating CD4+CD25- T cells under Th0 or Th17 conditions in the presence or absence of 1,25(OH)2D3. A) Suppressor cells were cultured with autologous DCs and antiCD3 for 24 hours with or without CTLA-4 blocking antibody. Expression of CD80, CD86, CD11c and CD40 by DCs was measured by flow cytometry. Dot plots show the ratio of marker expression in control versus anti-CTLA-4-treated cultures for four donors. B) Suppressor cells were CFDA-SE labeled and added to autologous DC plus antiCD3 stimulations of allogeneic cell trace violet labeled CD4+CD25- T cells (responders) with or without anti-CTLA-4. Parallel stimulations were also prepared in which CFDA-SE-labeled CD4+CD25- were added in place of suppressor T cells as a control for cell number. At five days, proliferation of responder T cells was assessed by flow cytometry. Data are from one donor but representative of four. Shaded histograms show proliferation in the absence of suppressors. Dotted and solid lines indicate proliferation in the presence versus the absence of anti-CTLA-4 respectively. Fig 6. 1,25(OH)2D3 promotes CTLA-4-mediated B7 depletion from dendritic cells and suppression of T Fig 6. Discussion A prominent feature of many autoimmune diseases is the inappropriate activation of T cells. T cell proliferation is initiated following TCR stimulation and supported by engagement of co- stimulatory CD28 with its ligands CD80 and CD86. Signals through CD28 are important for initiating and maintaining T cell proliferation, since they can overcome the induction of anergy [39]. How regulation through the CD28 axis is altered in settings of autoimmunity is therefore an important area of study. Expression of CTLA-4 is a major mechanism by which Treg cells elicit tolerance and the levels of CTLA-4 are higher in Foxp3 expressing T cells than in conven- tional activated T cells [40–42]. Nonetheless, expression of CTLA-4 by activated effector T 12 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0131539 July 2, 2015 Regulation of CTLA-4 by Vitamin D and Inflammatory Cytokines cells may also be important for some aspects of immune homeostasis. Indeed, the role of CTLA-4 in non-Tregs has recently been highlighted by two studies, which reveal a suppressive capability for activated T cells in-vivo [43, 44]. Altered expression of CTLA-4 by effector T cells could therefore affect the duration of a T cell response. In line with this, there are differences in disease kinetics between mice that lack CTLA-4 completely and those where CTLA-4 is defi- cient only in Treg [42, 45, 46]. In view of these studies it is of interest to understand how CTLA-4 is regulated. It is widely recognised that Th17 cells are key players in the development of many inflam- matory disorders [18–20]. Thus we were interested in how Th17 polarising conditions would affect CTLA-4 expression and function. We observed that a pro-Th17 cytokine cocktail of TGFβ, IL-1β, IL-6 and IL-23 reduced CTLA-4 expression in CD4+CD25- T cells after activa- tion and impaired their ability to remove co-stimulatory ligands by transendocytosis. Notably, this suppression of CTLA-4 was not restricted to Th17 cells but was evident across T cell classes including Foxp3+ in-vitro induced Tregs as well as T cells expressing IFNγ, IL-21, TNFα and IL-2. Thus suppression of CTLA-4 by pro-Th17 cytokines appears a general phenomenon that is not a consequence of the Th17 differentiation program. Physiologically, inflammation- driven down-regulation of CTLA-4 might be important to prolong T cell proliferation. Whilst this effect might be required for the efficient clearance of certain pathogens, down regulation of CTLA-4 at autoinflammatory sites could contribute to the persistence of the disease. Discussion In support of this hypothesis and our finding that Th17 conditions suppress CTLA-4 in FoxP3+ as well as FoxP3- T cells, a number of in-vivo studies have reported reduced CTLA-4 expression on Tregs and non-Tregs from patients with inflammatory disease [47–49]. Most importantly, their lack of CTLA-4 accounted for their loss of suppressive function [47]. Epidemiological studies suggest an inverse relationship between vitamin D status and the incidence of autoimmune diseases (reviewed in [25, 26, 29]). The significance of vitamin D in immune regulation is supported by the fact that immune cells, especially antigen presenting cells, express the enzyme 1α-hydroxylase that is necessary for vitamin D activation [50–53]. Indeed we have shown that activated T cells can induce 1α-hydroxylase activity in DCs at a level sufficient to influence the T cell response [51]. It has long been considered that the princi- pal mechanism by which 1,25(OH)2D3 suppresses T cell responses is by down-regulating MHC and co-stimulatory molecules on APCs [54–56]. However, our previous finding that 1,25(OH)2D3 increases CTLA-4 through direct effects upon the T cell, suggested that the sup- pressive effect of 1,25(OH)2D3 upon T cell activation might be further enhanced through CTLA-4-mediated removal of CD80/86 by transendocytosis. Through this study we have con- firmed that by increasing CTLA-4, 1,25(OH)2D3 can increase transendocytic removal of CD80/86 from APCs and substantially reduce their ability to stimulate T cells. Thus, 1,25 (OH)2D3 appears to target both the APC and T cell sides of the CD80/CD86-CD28/CTLA-4 axis to achieve maximal suppression of T cell responses. Importantly we have also shown that this ability of 1,25(OH)2D3 to increase CTLA-4 expression and transendocytic function is retained, if not enhanced, under Th17 polarising conditions. Taken together these data suggest that low levels of vitamin D might predispose to exaggerated inflammatory reactions along with impaired CTLA-4 expression and that provision of 1,25(OH)2D3 would reverse this effect even at sites of active Th17-driven inflammation. Many chronic inflammatory diseases develop as a consequence of exposure to environmen- tal triggers in a genetically susceptible individual. Interestingly, a number of polymorphisms associated with chronic inflammatory diseases lie within the co-stimulation gene locus at chro- mosome 2q33 that encodes CD28, CTLA-4 and ICOS [57–61] and ChIP-seq studies have revealed the presence of VDR binding sites near to these polymorphisms [62]. Discussion Thus regulation 13 / 18 PLOS ONE \| DOI:10.1371/journal.pone.0131539 July 2, 2015 Regulation of CTLA-4 by Vitamin D and Inflammatory Cytokines of CTLA-4 expression could be a point at which genetic and environmental factors interact to influence an immune response and the establishment of disease. Overall, our study has provided new insights into the regulation of CTLA-4, a critical sup- pressive protein whose loss of function is associated with autoimmunity. In particular we have shown that inflammatory Th17 conditions, suppress the expression and transendocytic func- tion of CTLA-4, but demonstrate that their effect can be opposed by 1,25(OH)2D3. In addition, we confirmed that the ability of 1,25(OH)2D3 to suppress inflammatory cytokines and increase IL-10 was retained even under inflammatory conditions. Our findings therefore highlight a potential role for vitamin D as an environmental factor in the control of autoimmunity and support the exploration of its use as a therapeutic agent for established disease as well as in prophylaxis. S2 Table. Summary of within subject effects and contrasts as determined by repeated mea- sure single factor within subject analysis for regulatory and inflammatory associated T cell markers. (PDF) S2 Table. Summary of within subject effects and contrasts as determined by repeated mea- sure single factor within subject analysis for regulatory and inflammatory associated T cell markers. (PDF) Author Contributions Conceived and designed the experiments: LEJ OSQ DG TH ZB BS JB KR DS. Performed the experiments: LEJ OSQ DG TH ZB BS JB. Analyzed the data: LEJ. Contributed reagents/materi- als/analysis tools: KR DS. Wrote the paper: LEJ OSQ DG TH ZB BS JB KR DS. Acknowledgments We thank Prof Martin Hewison for helpful discussions and Dr Peter Nightingale for statistical advice. S1 Table. Summary of within subject effects and contrasts as determined by repeated mea- sure two factor within subject analysis for regulatory and inflammatory associated T cell markers. (PDF) S1 Table. Summary of within subject effects and contrasts as determined by repeated mea- sure two factor within subject analysis for regulatory and inflammatory associated T cell markers. (PDF) S2 Table. Summary of within subject effects and contrasts as determined by repeated mea- sure single factor within subject analysis for regulatory and inflammatory associated T cell markers. (PDF) Supporting Information S1 Fig. Effect of Th17 polarising cytokines upon CTLA-4 expression. Cell trace-labelled CD4+CD25- T cells were stimulated for four days with antiCD3CD28 beads in the presence of recombinant cytokines TGFβ, IL-1β, IL-6 and IL-23 as indicated and expression of total CTLA-4 assessed by flow cytometry. Expression is given relative to the level in non-treated T cells. (TIF) S1 Table. Summary of within subject effects and contrasts as determined by repeated mea- sure two factor within subject analysis for regulatory and inflammatory associated T cell markers. (PDF) S2 Table. Summary of within subject effects and contrasts as determined by repeated mea- sure single factor within subject analysis for regulatory and inflammatory associated T cell markers. (PDF) PLOS ONE \| DOI:10.1371/journal.pone.0131539 July 2, 2015 References 2002; 16(1):23–35. PMID: 11825563. 13. Linsley PS, Bradshaw J, Greene J, Peach R, Bennett KL, Mittler RS. Intracellular trafficking of CTLA-4 and focal localization towards sites of TCR engagement. Immunity. 1996; 4(6):535–43. 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https://openalex.org/W1591165170	https://europepmc.org/articles/pmc4027891?pdf=render	English	null	ecancermedicalscience	ecancermedicalscience	2,016	cc-by	4,785	parna Gangopadhyay1, Joydeep Das1, Partha Nath2 and Jaydip Biswas2 Aparna Gangopadhyay1, Joydeep Das1, Partha Nath2 and Jaydip Biswas2 1Department of Radiation Oncology Chittaranjan National Cancer Institute, 37 SP Mukherjee Road, Kolkata 700026, India 2Department of Medical Oncology, Chittaranjan National Cancer Institute, 37 SP Mukherjee Road, Kolkata 700026, India Correspondence to: Aparna Gangopadhyay. Email: mails7778@gmail.com Abstract Background: Haemoglobin levels and tissue oxygenation influence tumour outcome in carcinoma cervix radiotherapy. The clinical impact of haemoglobin levels on acute normal tissue toxicity during radiation and interaction with chemotherapy in carcinoma of the cervix is underexplored. This paper aims to explore this issue. Methods: Treatment toxicity among 227 patients with squamous cell carcinoma of the cervix stages II B–IV A, receiving pelvic radiotherapy or chemoradiation at our institute, were studied prospectively. The baseline and weekly haemoglobin levels during treatment were recorded. Acute toxicities were recorded using Radiation Therapy Oncology Group (RTOG) acute toxicity and Common Terminology Criteria for Adverse Events (CTCAE) criteria, version 4. For the analysis, patients were divided into two groups, depending on nadir haemoglobin levels. A cut-off value for anaemia was selected at 12 gm/dL. Toxicity was compared between anaemic and non-anaemic groups. Results: Patients on chemoradiation and having haemoglobin levels >12 gm/dL suffered significantly higher dermatitis (two-tailed p value = 0.0288) and vaginal mucositis (two-tailed p value = 0.0187) of at least RTOG acute toxicity grade 2, compared with the anaemic group. In contrast patients receiving radiotherapy alone did not experience any significantly greater mucocutaneous toxicity if haemoglobin was >12 gm/dL. Anaemia had significantly greater impact on malaise and neutropenia (two-tailed p value <0.0001) of CTCAE grade 1 and above among chemoradiation patients, as opposed to those receiving radiotherapy alone (two-tailed p values = 0.0012 for neutropenia and 0.0422 for malaise). Conclusion: Haemoglobin values >12 gm/dL significantly worsen acute mucocutaneous toxicity in locally advanced cervical cancer patients receiving chemoradiation. Similar effects are not observed in the absence of chemotherapy. Keywords: cervix cancer, chemoradiation, haemoglobin levels, radiobiology Received: 09/03/2014 Published: 19/05/2014 Published: 19/05/2014 Published: 19/05/2014 ecancer 2014, 8:431 DOI: 10.3332/ecancer.2014.431 ecancer 2014, 8:431 DOI: 10.3332/ecancer.2014.431 Copyright: © the authors; licensee ecancermedicalscience. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1 1 ecancer 2014, 8:431 ecancer 2014, 8:431 Patients A total of 227 patients with locally advanced squamous cell carcinoma of the cervix, ranging from stages II B to IV A (Table 1), who received either pelvic radiotherapy or pelvic chemoradiation in the Department of Radiation Oncology at our institute from October 2010 to November 2012, were assessed for incidence of acute treatment toxicity during pelvic chemoradiation and HDR brachytherapy. Haemoglobin levels were routinely monitored during the same period. Introduction Carcinoma of the cervix is a common cancer in women in developing countries, usually presenting at advanced stages where surgery is no longer a treatment option [1]. As a consequence, radiotherapy is the mainstay in the treatment of these patients. From our perspective, a major health problem prevalent in the community is that of undernutrition, with anaemia being widely prevalent in the population. In India, the National Family Health Survey Data show that 55% of women of childbearing age are anaemic [2]. The World Health Organisation defines a value of 12 gm/dL as the cut-off value for anaemia in non-pregnant females above 15 years [3]. Although anaemia is defined variably in different oncology studies, 12 gm/dL was chosen as the cut-off point for anaemia in this paper to ensure that patients who were being deemed anaemic for the purposes of this paper were uniformly considered anaemic by National Community Health Programmes that follow WHO guidelines. An estimated third of carcinoma of the cervix patients at our institute have haemoglobin levels below 12 gm/dL at presentation, prior to start of treatment. It is well known that anaemia is an adverse prognostic factor for disease outcomes in locally advanced carcinoma of the cervix, due to the association between anaemia and tissue hypoxia, which translates to impaired tumour radiation effect. However, the impact of anaemia on normal tissue acute toxicity and, therefore, treatment compliance among patients with carcinoma of the cervix remains an area of investigation. The standard of care in locally advanced cervical carcinoma is pelvic chemoradiation. This treatment entails toxicity and may prove to be considerably morbid, especially when treatment volumes are large. Conformal radiotherapy techniques that help in reducing radiotherapy toxicity considerably cannot be offered to most patients in a limited-resources scenario. The problems of undernutrition, anaemia, a large volume of disease, and intensive treatment, all in the setting of limited resources, continue to remain a widespread challenge for patients and health-care staff. Under these circumstances, it was our endeavour to assess the impact of haemoglobin levels on acute treatment toxicity and treatment tolerance in these patients in our setting. The impact of haemoglobin levels on treatment toxicity was particularly relevant because it is a factor that may be easily corrected with appropriate interventions. Concomitant chemoradiation During the period of pelvic radiotherapy, all patients who had adequate creatinine clearance received weekly concomitant Cisplatin chemotherapy at a dose of 40 mg/m2 weekly. During the period of pelvic radiotherapy, all patients who had adequate creatinine clearance received weekly concomitant Cisplatin chemotherapy at a dose of 40 mg/m2 weekly. Any patient with contraindications to platinum therapy or those unwilling to receive chemotherapy received radiotherapy alone. Any patient with contraindications to platinum therapy or those unwilling to receive chemotherapy r Any patient with contraindications to platinum therapy or those unwilling to receive chemotherapy received radiotherapy alone. Pelvic radiotherapy Only those patients treated by conventional radiotherapy fields in telecobalt were studied to exclude any differences in toxicity that occur due to conformal planning or use of photons of variable energy. Brachytherapy Intracavitary HDR brachytherapy (Ir192) sequentially followed external beam radiotherapy was received by all patients. A dose of 7 Gy x 4 fractions was employed. External beam radiotherapy All patients received 50 Gy in 25 fractions five days a week to the pelvic field by telecobalt. Pelvic radiotherapy was planned conventionally in all studied patients. As per institutional protocol, anterior-posterior and posterior-anterior portals were employed. All patients received 50 Gy in 25 fractions five days a week to the pelvic field by telecobalt. Pelvic radiotherapy was planned conventionally in all studied patients. As per institutional protocol, anterior-posterior and posterior-anterior portals were employed. Methods Research Treatment Pelvic radiotherapy Methods The haemoglobin levels in all patients were recorded at the start of treatment and every week until completion of pelvic chemoradiation or radiotherapy. None of the patients had baseline haemoglobin levels below 10 gm/dL. Any patient needing transfusion for low haemoglobin levels after the start of treatment were excluded from the analysis. A total of 227 patients had been included in the study, of which 17 patients required blood transfusion during the course of treatment. These patients were excluded from analysis. The data of the remaining 210 patients were analysed (Table 2). As per institutional protocol, all patients had haemoglobin values of 10 gm/dL or above during the entire treatment period. nal protocol, all patients had haemoglobin values of 10 gm/dL or above during the entire treatment pe As per institutional protocol, all patients had haemoglobin values of 10 gm/dL or above during the en Treatment toxicity was assessed weekly for the entire period of external beam radiotherapy and also at first brachytherapy appointment using Radiation Therapy Oncology Group (RTOG) acute toxicity criteria and Common Terminology Criteria for Adverse Events (CTCAE) version 4.02. 2 2 www.ecancer.org ecancer 2014, 8:431 Table 1. Composition of study patient population according to stage, age, and histology. Stage Total number of patients Age ( in yrs) Histology 30–40 41–50 51–60 61–70 Well diff squamous cell carcinoma Mod diff squamous cell carcinoma Poorly diff squamous cell carcinoma II B 29 6 10 11 2 2 26 1 III A 8 3 3 2 0 0 5 3 III B 178 46 69 54 9 9 146 23 IV A 12 1 5 2 4 0 8 4 Table 1. Composition of study patient population according to stage, age, and histology. Table 2. Distribution of patients receiving either treatment modality according to disease stage. Stage Patients who received pelvic radiotherapy n = 86 Patients who received pelvic chemoradiation n = 124 II B 16 8 III A 0 2 III B 68 110 IV A 2 4 Table 2. Distribution of patients receiving either treatment modality according to disease stage. During data analysis, the nadir haemoglobin levels for each patient were taken into consideration, and incidence of acute treatment toxicity in relation to nadir haemoglobin levels was analysed. During data analysis, the nadir haemoglobin levels for each patient were taken into consideration, and incidence of acute treatment toxicity in relation to nadir haemoglobin levels was analysed. During data analysis, the nadir haemoglobin levels for each patient were taken into consideration, and incidence of acute treatment toxicity in relation to nadir haemoglobin levels was analysed. Results The ages of the patients receiving radiotherapy alone ranged from 39 to 68 years (median 54 years), and the group that received chemoradiation had a median age of 48 years (range 36–61 years). Overall, the commonest stage at presentation was III B, with 78.41% of the patients having stage III B disease at presentation. Stage III B also comprised 88.70% of chemoradiation patients and 79.06% of those who received only radiotherapy. Moderately, differentiated squamous cell carcinoma was the commonest histological diagnosis comprising of 81.5% of the study population (Table 1). Among 210 patients, 86 patients had received pelvic radiotherapy alone owing to comorbidities that prevented use of Cisplatin or refusing chemotherapy, and 124 patients received concurrent chemoradiation to the pelvis. Eighty-two patients had haemoglobin level of 12 gm/dL or higher. A total of 128 had haemoglobin levels between 10 and 12 gm/dL at baseline or at any time during therapy and comprised the anaemic group. Grade 1 or 2 dermatitis or vaginal mucositis was more common among patients who received chemoradiation and had a haemoglobin level of >12 gm/dL (45% with haemoglobin >12 versus 29% and 66% versus 33%, respectively), but not in patients who only received radiation (35% versus 29% and 50% versus 59%, respectively). As expected, patients with lower haemoglobin reported more grade 1 or 2 malaise than patients with haemoglobin >12 gm/dL. Of patients treated with chemoradiation, 79% of those with a haemoglobin <12 reported grade 1 or 2 malaise versus 21% of those with a higher haemoglobin. Of patients treated with radiation only, 35% of those with a haemoglobin <12 reported grade 1 or 2 malaise versus 8% of those with higher a haemoglobin level. Table 3. Occurrence of acute toxicity among patients from either treatment group. Toxicity assessment A weekly assessment of acute treatment-related toxicity based on RTOG acute toxicity criteria was made in all patients with respect to genitourinary and lower GI symptoms, dermatitis, and vaginal mucositis, although the latter is not easily amenable for assessment during treatment. In addition, neutropenia, vomiting, anorexia, malaise, urinary tract infection, and pelvic infection were assessed and graded weekly according to CTCAE version 4.02. For all patients, the highest grade of acute toxicity as per the RTOG scoring criteria or CTCAE grade suffered during treatment was taken into consideration for analysis. 3 www.ecancer.org ecancer 2014, 8:431 ecancer 2014, 8:431 ecancer 2014, 8:431 ecancer 2014, 8:431 Statistical analysis For analysis, patients were divided into two groups, depending on whether they were anaemic or non-anaemic. A cut-off value was selected at 12 gm/dL in accordance with the WHO definition of anaemia [4]. Any patient with nadir haemoglobin levels of 12 gm/dL or above was considered non-anaemic. All other patients were included in the anaemic group. Fisher’s t-test (two sided) was used to assess any significant differences in acute treatment toxicity between anaemic and non-anaemic groups receiving either treatment modality (Table 3). Statistical analysis was done using the Graph Pad QuickCalcs Web site: http://www.graphpad.com/quickcalcs/ contingency1 (accessed June 2013). Statistical analysis was done using the Graph Pad QuickCalcs Web site: http://www.graphpad.com June 2013). Results he incidence of urinary tract infection was significantly higher in anaemic patients who received chemora rinary tract infection was significantly higher in anaemic patients who received chemoradiation (two-taile The incidence of urinary tract infection was significantly higher in anaemic patients who received chemoradiation (two tailed p value 0.0207). Treatment delays were higher in anaemic patients receiving chemoradiation, but statistical significance was not noted. Treatment delays were higher in anaemic patients receiving chemoradiation, but statistical significance was not noted. reatment delays were higher in anaemic patients receiving chemoradiation, but statistical significanc were higher in anaemic patients receiving chemoradiation, but statistical significance was not noted. Among patients who received radiotherapy alone, there was no significant difference between acute radiation dermatitis and vaginal mucositis of grade 2 or lower experienced by patients who were anaemic as against those who had haemoglobin levels of 12 gm/dL or higher. However, similar to the chemoradiation group, neutropenia was significantly higher in the anaemic patients receiving radiotherapy alone (two-tailed p value = 0.0012). Malaise was also significantly higher in anaemic patients who received radiotherapy alone (two-tailed p value = 0.0422), although the difference was not as pronounced as those who received chemoradiation. However, similar to the chemoradiation group, neutropenia was significantly higher in the anaemic patients receiving radiotherapy alone (two-tailed p value = 0.0012). Malaise was also significantly higher in anaemic patients who received radiotherapy alone (two-tailed p value = 0.0422), although the difference was not as pronounced as those who received chemoradiation. Considering the anaemic patients from both treatment groups separately, there was no significant difference between treatment toxicity depending on whether anaemia was at baseline or at any time during therapy (Table 4). Considering the anaemic patients from both treatment groups separately, there was no significant difference between treatment toxicity depending on whether anaemia was at baseline or at any time during therapy (Table 4). On completion of treatment, a clinical response was found in 44/48 (91.7%) anaemic patients who received chemoradiation as opposed to 71/76 (93.4%) non-anaemic patients who had received the same treatment. This was found to be statistically non-significant. A ti t h i d di th l li i l t i i ti t d i ti t 22/34 Among patients who received radiotherapy alone, clinical response rates in anaemic patients and non-anaemic patients were 22/34 (64.7%) and 35/52 (67.3%), respectively, which was also not statistically significant. Table 4. Results Toxicity during treatment Pelvic radiotherapy (n = 86) p-values Pelvic chemoradiation (n = 124) p-values Anaemic (n = 34) Non-anaemic (n = 52) Anaemic (n = 48) Non-anaemic (n = 76) Dermatitis 10 29% 18 35% NS 14 29% 34 45% 0.0288 Mucositis 20 59% 26 50% NS 16 33% 50 66% 0.0187 Genitourinary 2 6% 0 0% NS 4 8% 2 3% NS Lower gastrointestinal 4 12% 2 4% NS 2 4% 0 0% NS Neutropenia 16 47% 2 4% 0.0012 30 63% 8 11% <0.0001 Nausea 0 0% 0 0% NS 34 70% 38 50% NS Vomiting 0 0% 0 0% NS 4 8% 2 3% NS Anorexia 6 17% 4 8% NS 30 62% 32 42% NS Malaise 12 35% 4 8% 0.0422 38 79% 16 21% <0.0001 Urinary tract infection 0 0% 2 4% NS 4 8% 0 0% 0.0207 Pelvic infection 2 6% 0 0% NS 0 0% 0 0% NS NS = Not significant. able 3. Occurrence of acute toxicity among patients from either treatment group. 4 www.ecancer.org ecancer 2014, 8:431 ecancer 2014, 8:431 Grade 1 or 2 neutropenia likewise was more common among patients with lower haemoglobin level. Sixty-three per cent of patients who received chemoradiation and had a nadir haemoglobin level of <12 gm/dL also developed grade 1 or 2 neutropenia during therapy versus 11% of those with a higher haemoglobin level. Forty-seven per cent of patients who only received only radiation and had a nadir haemoglobin level <12 developed grade 1 or 2 neutropenia versus 4% of those with higher haemoglobin. Significance was tested for the association of these toxicities and haemoglobin levels and compared by treatment modality. For patients receiving chemoradiation, the group having haemoglobin levels higher than 12 gm/dL suffered significantly higher incidence of dermatitis (two-tailed p value = 0.0288) and vaginal mucositis (two-tailed p value = 0.0187) of RTOG acute toxicity grade 2 or lower, compared with the anaemic group receiving same treatment. None of the patients in either group experienced grade 3 or 4 toxicity. There was very significant difference in the incidence of neutropenia and malaise between both groups. Patients who were anaemic experienced far more malaise (two-tailed p value <0.0001) and neutropenia (two-tailed p value <0.0001) of CTCAE grade 1 and above compared with patients who were not anaemic. No toxicity of grade 3 or 4 was noted. Discussion Iron deficiency anaemia is the most prevalent nutritional deficiency among Indian women. It is already established that anaemia is an adverse prognostic factor for treatment response among patients with carcinoma of the cervix receiving concurrent chemoradiation or radiotherapy alone [5]–[7]. Literature also points to worse outcomes among cervix cancer patients who require blood transfusions during treatment [8]. Unfortunately, anaemia and cervix cancer are both significant problems in women’s health care in our scenario, with quite a few patients requiring transfusions to maintain adequate haemoglobin levels during treatment. While it is established that anaemia adversely impacts tumour outcomes during radiotherapy for carcinoma of the cervix, any impact of anaemia on normal tissue outcome is hardly available in literature for these patients. While it is established that anaemia adversely impacts tumour outcomes during radiotherapy for c anaemia on normal tissue outcome is hardly available in literature for these patients. Sufficient data in this regard from this part of the world, where both anaemia and cervix carcinoma are major health problems among women, are also not widely available. There is evidence in the literature, mostly from other tumours, that anaemia influences acute radiation toxicity owing to the effect of hypoxia on acutely reacting normal tissue. A number of clinical studies that reported radiation therapy outcomes in relation to anaemia have not shown consistent results, indicating the need for further exploration. A number of clinical studies that reported radiation therapy outcomes in relation to anaemia have n the need for further exploration. Henke et al [9] demonstrated lower acute radiation toxicity in anaemic subjects among a group of head and neck cancer patients receiving radiotherapy. The authors concluded that tissue hypoxemia could be the reason for less-severe acute reactions in anaemic patients. It is known that anaemia has adverse prognostic impact on tumour outcome in head and neck cancer radiotherapy similar to cervical cancer; whether the effects in relation to normal tissue toxicity for cervix cancer patients are similar is an area of interest. Daly et al [10] studied acute radiation toxicity in locally advanced head and neck cancer patients treated by two different fractionation schedules. Normal tissue toxicity, as defined by the incidence of confluent mucositis was assessed and compared between two patient groups, one having anaemia and the other group without anaemia. Results Toxicity differences among anaemic patients, depending on baseline anaemia or at anytime during treatment. Toxicity during treatment among anaemic patients (either at baseline or any time during therapy) Pelvic radiotherapy Pelvic chemoradiation Total (n = 34) Anaemia at baseline (n = 20) Anaemia at any time during treatment (n = 14) Total (n = 48) Anaemia at baseline (n = 29) Anaemia at any time during treatment (n = 19) Dermatitis 10 6 4 14 5 9 Mucositis 20 8 12 16 9 7 Genitourinary 2 0 2 4 1 3 Lower gastrointestinal 4 2 2 2 0 2 Neutropenia 16 11 5 30 14 16 Nausea 0 0 0 34 11 23 Vomiting 0 0 0 4 3 1 Anorexia 6 2 4 30 9 21 Malaise 12 7 5 38 20 18 Urinary tract infection 0 0 0 4 2 2 Pelvic infection 2 1 1 0 0 0 s among anaemic patients, depending on baseline anaemia or at anytime during treatment. 5 5 www.ecancer.org ecancer 2014, 8:431 ecancer 2014, 8:431 The presence of anaemia according to the cut-off values chosen for this paper did not seem to affect disease response significantly. The presence of anaemia according to the cut-off values chosen for this paper did not seem to affec Unfortunately, many patients studied had attended local hospitals and did not attend our institute for follow up in the long term, so it was not possible to assess for impact on late toxicity and recurrence patterns in this paper population. It is relevant to state that a choice of a different and lower cut-off value for anaemia would possibly have yielded different results. oice of a different and lower cut-off value for anaemia would possibly have yielded different results. Conclusion Radiation therapy aims at optimising therapeutic ratio by improving tumour cell kill and simultaneously reducing normal tissue toxicity. Widely exploited in the clinic is the knowledge of the radiobiology of different tissues, as well as use of agents that modify tumour response. The mechanisms of the interplay of factors that modify radiation effect are complex, and may lead to variable clinical outcomes. In the case of carcinoma of the cervix, there is established evidence that anaemia and hence hypoxia and radiosensitizers both have significant impact on tumour response. The normal tissue impact of the interaction between these factors has been relatively underexplored, although it is equally relevant in terms of clinical outcomes. It was the aim of this study to explore the differences in normal tissue toxicity between groups of patients during treatment who had differences in two factors, chemotherapy and anaemia. The results from this study showed that the interaction of anaemia, and hence hypoxia with chemotherapy reduced acute radiation toxicity selectively in skin and vaginal mucosa, although an accurate estimate of vaginal mucositis during treatment is not easily made. Similar effects were not observed for other organs. This paper results cannot be generalised owing to limitations in regard to patient number, treatment technique, the use of chemotherapy as the only radiosensitizer and also defining anaemia at a chosen cut-off value. Whether these results would vary for a larger cohort, for those receiving conformal radiotherapy and use of alternative radiosensitizers, warrants further investigation. Furthermore, this paper focused on acute toxicity only. Studies are needed to assess any difference in late effects and their clinical impact, if any. Discussion The results showed no significant differences in normal tissue toxicity between both patient groups, although disease control was inferior among anaemic patients. Their study demonstrated absence of any difference in normal tissue toxicity due to anaemia. In either of these studies on head and neck patients, chemotherapy was not added; the interaction between chemotherapy and anaemia could not be commented upon by the authors. A study involving a large patient cohort from Spain, predominantly comprising breast cancer patients, explored the possible relationship between haemoglobin levels and incidence of acute radiation toxicity. This cohort comprised of patients who were treated by conventional radiotherapy and some patients had received concomitant chemotherapy, too. The correlation between absolute haemoglobin values and the degree of toxicity, and the possible relationship between haemoglobin concentration and the appearance of toxicity due to radiotherapy was not found to be significant. Differences in toxicity based on addition of chemotherapy or variable radiotherapy technique were not reported [11]. Radiosensitizers such as chemotherapy that improve therapeutic ratio by enhancing tumour control may potentially alter normal tissue toxicity. When these are combined with the added effect of hypoxia, it is possible that the interplay of these factor scould affect normal tissue toxicity in addition to tumour. 6 6 www.ecancer.org ecancer 2014, 8:431 ecancer 2014, 8:431 The discovery of the hypoxia inducible factor-1 alpha (HIF-1α) and its role in tumour behaviour and treatment response has increased interest in the signal molecules related to tumour hypoxia. Recent evidence suggests that radio sensitization does not just involve compounds that enhance the effect of radiation by mimicking oxygen, but explores compounds that target HIF-mediated signaling pathways eventually reducing radio resistance [12]. Considering that radiosensitizers have diverse mechanisms of action and that they may interact variably with hypoxia, a huge number of probabilities are thrown open in regard to tumour and normal tissue behaviour. In addition, differences in radiobiology of different tissues further widen probabilities. The interesting finding in our patients was that patients with haemoglobin levels of 12 gm/dL or greater (N = 38) who received chemoradiation had more grade 1 or 2 dermatitis or vaginal mucositis than those with lower haemoglobin level (N = 24). This association was not seen in absence of Cisplatin. Evidently, hypoxia reduced normal tissue effects of Cisplatin. In our study group most patients had conventional treatment fields. Conflicts of interest The authors have no conflicts of interest to declare. Discussion Conformal radiation techniques that improve therapeutic ratio of radiotherapy treatment play a prominent role in cervix cancer owing to proximity of target volume with organs at risk. However, the potential for greater normal tissue effects due to higher integral dose in the case of intensity-modulated radiotherapy also exists. The normal tissue effects in treatments with higher integral dose may differ from one individual to another on the basis of these interactions between tissue oxygenation and radiosensitizers. 1. Ferlay J, Soerjomataram I, Ervik M, et al (2013) GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC Cancer Base No. 11 [Internet]. (Lyon, France: International Agency for Research on Cancer) Available from: http://globocan.iarc.fr. Last accessed December 2013. 2. International Institute for Population Sciences (IIPS) and Macro International (2007) National Family Health Survey (NFHS-3), 2005–06: India: Volume I (Mumbai: IIPS) References 1. Ferlay J, Soerjomataram I, Ervik M, et al (2013) GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC Cancer Base No. 11 [Internet]. (Lyon, France: International Agency for Research on Cancer) Available from: http://globocan.iarc.fr. Last accessed December 2013. 2. International Institute for Population Sciences (IIPS) and Macro International (2007) National Family Health Survey (NFHS-3), 2005–06: India: Volume I (Mumbai: IIPS) 7 7 www.ecancer.org ecancer 2014, 8:431 ecancer 2014, 8:431 3. WHO (2011) Haemoglobin concentrations for the diagnosis of anaemia and assessment of severity. Vitamin and Mineral Nutrition Information System. (Geneva: World Health Organization) (WHO/NMH/NHD/MNM/11.1) (http://www.who.int/vmnis/indica- tors/haemoglobin.pdf, accessed December 2013). 4. Available from http://www.who.int/vmnis/indicators/haemoglobin.pdf, accessed June 2013 5. Obermair A et al (2003) Anemia before and during concurrent chemo radiotherapy in patients with cervical carcinoma: effect on progression-free survival Int J Gynecol Cancer 13(5) 633–9 DOI: 10.1046/j.1525-1438.2003.13395.x PMID: 14675347 5. Obermair A et al (2003) Anemia before and during concurrent chemo radiotherapy in patients with cervical carcinoma: effect on progression-free survival Int J Gynecol Cancer 13(5) 633–9 DOI: 10.1046/j.1525-1438.2003.13395.x PMID: 14675347 6. Dunst J et al (2003) Anemia in cervical cancers: impact on survival, patterns of relapse, and association with hypoxia and angiogenesis Int J Radiat Oncol Biol Phys 56(3) 778–87 Pub Med PMID: 12788185 6. Dunst J et al (2003) Anemia in cervical cancers: impact on survival, patterns of relapse, and association with hypoxia and angiogenesis Int J Radiat Oncol Biol Phys 56(3) 778–87 Pub Med PMID: 12788185 7. Yalman D et al (2003) Prognostic factors in definitive radiotherapy of uterine cervical cancer Eur J Gynaecol Oncol 24(3–4) 309–14 Pub Med PMID: 12807246 7. Yalman D et al (2003) Prognostic factors in definitive radiotherapy of uterine cervical cancer Eur J Gynaecol Oncol 24(3–4) 309–14 Pub Med PMID: 12807246 8. Santin AD et al (2003) Influence of allogeneic blood transfusion on clinical outcome during radiotherapy for cancer of the uterine cervix. Gynecol Obstet Invest 56(1) 28–34 Epub 2003 Jul 14 Pub Med DOI: 10.1159/000072328 PMID: 12867765 9. Henke M et al (2000) Blood hemoglobin level may affect radiosensitivity-preliminary results on acutely reacting normal tissues Int J Radiat Oncol Biol Phys 48(2) 339–45 DOI: 10.1016/S0360-3016(00)00595-2 PMID: 10974446 10. Daly T et al (2003) The effect of anaemia on efficacy and normal tissue toxicity following radiotherapy for locally advanced squamous cell carcinoma of the head and neck Radiother Oncol 68(2) 113–22 Pub Med DOI: 10.1016/S0167-8140(03)00198-1 PMID: 12972305 Research 11. References Escó Barón R et al (2005) Hemoglobin levels and acute radiotherapy-induced toxicity Tumori 91(1) 40–5 Pub Med PMID: 15850003 11. Escó Barón R et al (2005) Hemoglobin levels and acute radiotherapy-induced toxicity Tumori 91(1) 40–5 Pub Med PMID: 15850003 12. Ghattass K et al (2013) Targeting hypoxia for sensitization of tumors to radio- and chemotherapy Curr Cancer Drug Targets 13(6) 670–85 PubMed DOI: 10.2174/15680096113139990004 PMID: 23687923 12. Ghattass K et al (2013) Targeting hypoxia for sensitization of tumors to radio- and chemotherapy Curr Cancer Drug Targets 13(6) 670–85 PubMed DOI: 10.2174/15680096113139990004 PMID: 23687923 8 8 www.ecancer.org
W2128578024.txt	https://zenodo.org/records/1232285/files/article.pdf	en		Characterization of frequency stability in precision frequency sources	Proceedings of the IEEE	1,991	public-domain	6,934	Characterization of Frequency Stability In Precision Frequency Sources JACQUES RUTMAN, SENIOR MEMBER, IEEE, AND F. L. WALLS Invited Paper This paper presents a short review of the progress that has occurred over the past 25-30 years in both the theoretical and practical characterization of frequency stability of precision frequency sources. The emphasis is on the evolution of ideas and concepts for the characterization of random noise processes in such standards in the time domain and the Fourier frequency domain, rather than a rigorous mathematical treatment of the problem. Numerous references to the mathematical treatments are made. I. INTRODUCTION High precision frequency standards have undergone tremendous advances during the decades since the advent of the first laboratory cesium beam clock in 1955. Thousands of atomic clocks, such as the cesium beam and the optically pumped rubidium standards manufactured by industry, are in routine use today. The ultrastable hydrogen maser is also used on a large scale for very demanding applications. Quality quartz-crystal-controlled oscillators have also shown such progress in stability that they can sometimes compete with rubidium clocks. These devices are used in applications such as: fundamental metrology, telecommunications systems, space missions, radars, broadcasting, etc. By the early-1960’s it was clearly recognized that there was a real need for a common set of frequency stability characterization parameters and for related measurement test-sets [l], [2]. These parameters were needed for at least two main purposes: 1) to allow for meaningful comparisons between similar devices developed by different laboratories, or between different devices in a given application; 2) to access application performance in terms of the measured oscillator frequency stability. Manuscript received February 2, 1991; revised March 21, 1991. J. Rutman is with the Federation des Industries Electriques et Electroniques, 75116 Paris, France. F. L. Walls is with the Time and Frequency Division, National Institute of Standards and Technology, Boulder, CO 80303. IEEE Log Number 9101209. Related experimental test sets with well-defined characteristics were of course needed in order to unambiguously measure the various frequency stability parameters. This paper presents a short review of the progress that has occurred during the last 25-30 years both in terms of theoretical characterization of frequency stability and of experimental measurement test sets. 11. CHARCTERIZATION OF FREQUENCY STABILITY In this paper no attempt will be made to give mathematical developments that can be found in many references [1]-[47]. Reference [21] contains many of the original papers with errata sheets. It attempts to point out inconsistencies between the notation of these papers and the updated recommendations of IEEE [7] and CCIR [38]. We will concentrate mainly on the evolution of ideas and concepts and try to highlight the key milestones with a minimum of mathematical symbols and equations. In simple terms, the practical problem is how to characterize the properties of the output signal from a real oscillator. The output signal from an ideal noise-free nondrifting oscillator would be a pure sine wave, but any real device, even the most stable, is disturbed by unavoidable processes such as random noises, drifts due to aging andlor environmental effects. This paper will be mainly devoted to the characterization of frequency instabilities due to random noises which exist in all kinds of devices. Hence, the first step is to develop a tractable mathematical model for the quasi-sinusoidal output signal of an oscillator. MODEL 111. OUTPUTSIGNAL A relatively simple model that was introduced in the early 1960’s and has found wide acceptance is where $(t)is a random process denoting phase noise [6], [7], [21], [33], [37], [38], [43], VOand vo are the nominal amplitude and frequency respectively; and amplitude noise , U.S. Government Work Not Protected by U.S. Copyright PROCEEDINGS OF THE IEEE, VOL. 79, NO. 6, JUNE 1991 952 - characterized by &(t)that can usually be neglected in high performance sources. (In this treatment we assume that frequency drift, if any, has been removed.) Such a quasisinusoidal signal has an instantaneous frequency defined as + 1 d+(t) qqt))= vo 4-27r dt 21r dt * Frequency noise is the random process defined by 1 -(27rvot d v(t) = - (2) related by the simple law [6], [7], (211, [33], [37], [38], sAv(f) = f2s+(f) which corresponds to the time derivative between + ( t ) and Av(t).The spectral density Sy(f)is also widely used and is very simply related to S,,(f) and S@(f)by 1 Sdf) = --s vo” (3) which exists simultaneously with and has properties similar to phase noise, as will be seen later. Very often it is useful to introduce the normalized dimensionless frequency fluctuations, This quantity remains unchanged under frequency multiplication or division and can be used as a basis for comparisons of oscillators at different nominal frequencies. Since we have modeled phase and frequency fluctuations by random processes, we are now in a position to use the various statistical tools which allow fluctuation characterization, such as correlation functions, spectral densities, averages, standard deviations and variances etc. Many textbooks exist on this subject. Now the problem of frequency (or phase) instability characterization is to introduce meaningful and practical (i.e. measurable) parameters for describing the statistical properties of +(t),4 t ) ,Av(t), 01 d t ) . IV. THE GREATDICHOTOMY Users of frequency standards in various fields recognized early on that they needed two kinds of parameters in order to meet requirements of different applications-namely spectral parameters (related to the spread of signal energy in the Fourier frequency spectrum) and time parameters (allowing assessment of the stability over a given time interval). Therefore two sets of parameters have been introduced as tools for oscillator characterization: 1) spectral densities of phase and frequency fluctuations, in the so-called Fourier frequency domain. 2) variances (or standard-deviation) of the averaged frequency fluctuations in the time domain. We first view briefly these two different kinds of parameters and then describe the mathematical relationships between them together with the related experimental consequences. A key point is the integral relationship which allows us to derive the variances from the knowledge of the spectral densities. DOMAIN V. FOURIERFREQUENCY In the Fourier frequency domain, phase and frequency fluctuations can be characterized by the respective onesided spectral densities, S@(f ) and Sau(f), which are (5) A’ (f)= $S@(f). vo (6) (Note the word “frequency” is used with two different meanings which should not be confused. v(t)is the timedependent instantaneous frequency of the oscillator, and f is the time-independent Fourier frequency that appears in any spectral density. The spectral density is the Fourier transform of the autocorrelation function [6], [33].) It has been shown from both theoretical considerations and experimental measurements, that the spectral densities due to random noise of all high stability frequency standards can be modelled by the power law model where the spectral densities vary as a power of f . More specifically, Sy(f) can be written as the sum: (7) a=-2 for 0 5 f 5 fh where fh is an upper cutoff frequency. For a given type of oscillator two or three terms of the sum are usually dominant. Each term is related to a given noise source in the oscillator (internal and/or external white noise, flicker noise,. . .). The most common noise types encountered in practical sources are given in Table 1. Of course, power laws can sometimes lead to “mathematical pathologies” (divergence of integrals) when they are integrated from f = 0 to f = CO, but this is only a limitation of the model that can be overcome by physical considerations (limited bandwidth and duration, for example). Sy(f)was proposed in 1971 by the IEEE as the recommended frequency stability measure in the Fourier frequency domain [ 6 ] . The updated version given in [7] is in general agreement with the recommendations of CCIR [371, 1381. For stationary Gaussian random processes, the spectral density (or the autocorrelation function) contains the maximum information about the process. The variances that will be defined later are all related to the spectral densities via integrals and transfer functions. Some information is, however, lost in the process. Spectral densities of phase or frequency are measured by a spectrum analyzer (analog or fast Fourier transform) following some kind of demodulation of $(f) or Av(t). Numerous experimental tests sets have been developed for that purpose [8], [MI, [23], [39]. Figure 1 shows Sy(f) for the five common power-law noise processes listed in Table 1. Specific techniques for measuring S@(f)(or equivalently Sy(f))are described in [8], [9], [13], [18], [19], [21]-[23], [43]-[45]. Particular attention is focused on describing the errors in such measurements and in R U T M A N AND WALLS: FREQUENCY STABILITY IN PRECISION FREQUENCY SOURCES 953 Table 1 Listing of the five common types of noise found in practical sources. The asymptotic forms of two filter types are also listed. Note: W h / 2 ? r =fh is the measurement system bandwidth-often U$(.) for various power-law noise types and called the high-frequency cutoff. In E log u:(r) Whr Name of Noise * SY(f) >> 1 Infinite Sharp Filter WhT >> 1 WhT Single Pole Filter << 1 Infinite Sharp Filter WhT << 1 Single Pole Filter t f Fig. 1. Spectral density of frequency fluctuations S y ( f ) for the five wmmon noise types. determining S,(f) or S+(f)for a single oscillator in [8], [18], [23]. Confidence intervals for spectral density measurement of nonwhite noise are treated in [41]. VI. TIMEDOMAIN Time-domain characterization of frequency stability is widely used since it answers the obvious question: what is the stability over a time interval T for a given application? (T can range from milliseconds to months and years according to the application.) In the time domain, the basic measurement apparatus is a digital counter that yields results that can be related to vi, the ith average value of y(t) over a time interval r beginning at time ti (any physical measurement has a finite duration r that cannot approach zero; instantaneous frequency cannot be measured). Figure 2 shows the basic measurement cycle. Simple counting techniques are, however, severely limited in precision. Heterodyne techniques offer much higher accuracy at the expense of increased complexity [9], [20], [21], [24], [42]. In order to assess frequency stability over a time interval r (the sample time), it is necessary to make a series of measurements, each of duration T , which yields the results with i = 1-N. Of course, due to the random fluctuations of y(t), the vi's are samples of a random variable and frequency stability over r which can only be defined from a measure of the dispersion. A widely used statistical tool for that is the variance, u2,or the square root of the variance, u, normally called the standard deviation. Time Fig. 2. Measurements process for the computation of sample variances. The phase difference, between two oscillators, z ( t ) , is plotted as a function of time. Here T is the total cycle time and r is the averaging time for each measurement. The fractional frequency difference Y k is given by = z(tk+r!-z(tkl. vk A very specific problem for oscillator characterization is that several kinds of variances have been introduced by several authors since the early 1960's and thus it is necessary to give a clear picture of the relationships (if any) between the variances and the spectral densities. A. Tnre Variance The true variance is a theoretical parameter denoted as 12(.) and simply defined as: 12(r)=<$>. When y(t) has a zero mean, the bracket e > denotes an infinite time average made over one sample of y(t). For stationary frequency fluctuations around vo ,I2(T ) decreases from <y2(t)>for T = 0 to 12(r) = 0, for r + CO where fluctuations are completely averaged away as shown in curve a of Fig. 3. However, despite its mathematical simplicity, the true variance is not really useful for experimental purposes since it approaches infinity for all real oscillators as shown by curve b in Fig. 3. Practical estimators of the timedomain stability relying on the sample variance concept were introduced in 1966 to avoid the divergence of the true variance observed in most sources [5]. vi 954 B. Sample Variance The sample variance is a more practical estimate of timedomain stability based upon a finite number of N samples yk(k = 1-N) than the time variance. Each sample has a duration T , and the kth sample begins at t k ; the (k 1)th + PROCEEDINGS OF THE IEEE, VOL. 79, NO. 7, JULY 1991 there are no recommended values for the measurement bandwidth, fh, it has to be specified with any experimental results for comparison purposes (and also because the result can be fh-dependent for some kinds of noise.) ‘R D. Estimates of the Allan Variance Experimentally only estimates of o;(r)can be obtained from a finite number of samples gk taken over a finite duration. Therefore an inherent statistical uncertainty (error bars) exists when m values of g k are used to estimate o,”(r). A widely used estimator is: Log r 0 Fig. 3. Frequency instability in the time-domain. (a) Square root of the true variance for stationary frequency noise. @) Performance of practical frequency sources, i.e., reality. . sample begins at t k + 1 = t k +T; the dead time between two successive samples is then T - r. The quantity T is the repetition interval for individual measurements of duration r as shown in Fig. 2. The sample variance is defined as m-1 This quantity is itself a random variable whose variance (the variance of the variance) may be used to calculate ) 7. This subject the error bars on the plot of c ~ ( Tversus was treated in great detail by Lesage and Audoin in 1973, when they calculated the error bars for Gaussian noises characterized by power law spectral densities [ll]. An additional treatment of the confidence limits is to be found in [21]. For long-term stability (r of the order of days, months, or even a year) the size of m is severely limited. In any case, m should be stated with any results to avoid ambiguity and to allow for meaningful comparisons. Estimates of the bias in experimental measurements made with dead time have been made by Barnes [12], as shown below: This quantity is itself a random variable, N being the sample size. Its average can be used as a measure of frequency stability over a time interval ~ : < a i ( N T,, T ) > . (See [3] for more detailed considerations about the various possible definitions of sample variances and their respective advantages or limitations-biased or unbiased estimates.) C . Allan Variance With the sample variance as defined above, we are faced with several possible values for the parameters N and T (besides r , which is the duration over which stability is measured). To achieve the goal of recommending a unique time-domain measure that can be used unambiguously in laboratories all around the world, some choices must be made. Following the pioneering work of David Allan in 1966 [5], the IEEE subcommittee on frequency stability recommended in 1971 to use the average of the variance with N = 2 and adjacent samples (that is T = r, or zero dead time) [6]. The resulting measure is denoted as 1 o;(r) = - <(& 2 (9) and known as the Allan variance, or two-sample variance, since pairs of adjacent measurements are grouped together. oi (T ) is also a theoretical measure since infinite duration is implied in the average denoted as < >. However, it has a much greater practical utility than 12(.) since it exists for all the spectral density power laws encountered in real oscillators (Table 1) including flicker frequency noise. This will be shown later from the mathematical relationship between frequency- and time-domain parameters. Moreover, simple experimental estimates may be derived for a;(.) since groups of only two measurements are involved. The choice of N = 2 in the preferred stability time domain measure is really the key feature in the definition of oi(r).Although B2 is the bias function given in [12]. This is of practical interest since counting techniques usually have nonzero dead time between successive measurements. Estimates of the biases caused by unequally spaced data are given by Barnes and Allan [25]. Most precision measurement techniques eliminate the problems of dead time and hence do not require these bias functions [20], [24], [42]. Techniques for determining oY(7)for an individual oscillator are treated in [17], [NI, [21], [23], [29], [44] as well as many of the other references. Figure 4 shows the dependence of ay(.) on measurement time for the five common power-low noise process in the limit that 2TfhT is large compared to 1. E. Comments on U,(.) The slope of cy(.) versus 7 is virtually the same for a = 1 and a = 2. As a consequence oY(r)is not useful for distinguishing between these noise types. With both noise types, frequency (or phase) fluctuations at f = fh dominate o,(T), even for extremely long measurement times. Changes in the average frequency over long times do not bias the characterization of short-term frequency stability as occurs with 12(.). The determination of oy(.) is dependent on the noise bandwidth and, in the limit 27rfhT 5 1, the type of low-pass filter. This is illustrated for (Y = 2 in Fig. 5 . c;(T) is a very efficient estimator for noise types a = 0, -1, -2 but diverges for a 5 -3. RUTMAN AND WALLS. FREQUENCY STABILITY IN PRECISION FREQUENCY SOURCES -- 1--- ~ 955 ~~ ~ a ....................................... -.-. I 1 E+OO 1 1 1 1 1 I 1 1 1 Measurement Time (s) d- b I Fig. 6. Measurement process for determining xi's used in the determination of mod U,(.). e .?.= 1 E+03 1.E-09 Fig. 4. U',(.) versus r for the five common power-law noise types in the limit that 2TfhT is large compared to 1 and an infinitely sharp filter is used. Curve a is for random-walk frequency modulation, Sy(f) = h - ~ f - ~ C.urve b is for flicker frequency modulation, S,(f) = h-lf-'. Curve c is for white frequency modulation, S,(f) = ho. Curve d is for flicker phase modulation, S,(f) = hlf and a bandwidth of 16 Hz. Curve e is for white phase modulation, S,(f)= h 2 f 2 and a bandwidth of 16 Hz.h-2 = h-1 = ho = hl = h2 = 2 x 10-24. E- a Mod I.E-17F I 1.E+OO I I IIIIII I I I IIIIII Measurement Time (s) I I I llllll l.E+03 Fig. 7. Mod U , ( T ) versus T for the five common power-law noise types in the limit that 2TfhTO is large compared to 1 and an infinitely sharp filter is used. Curve a is for random walk frequency modulation, S,(f) = h - 2 f - I . Curve b is for flicker frequency modulation, S,(f) = L l f - . Curve c is for white frequency modulation, S,(f) = ho. Curve d is for flicker phase modulation, S,(f) = hl f and a bandwidth of 16 Hz. Curve e is for white phase modulation, S,(f) = h 2 f 2 and a bandwidth of 16 Hz. h-2 = h-1 = ho = hl = hz = 2 ~ 1 0SeeFig. ~ ~ 4forthevalues ~ . for the same noise levels. of U,(.) Fig. 5. u , ( T ) for white phase modulation (cy = 2) as a function of measurement time, T , and measurement bandwidth, fh. Curves a, b, and c have an infinitely sharp filter with width, f h = 16 Hz, fh = 0.016 Hz, f h = 0.0016 Hz, respectively. Curves d and e have a single pole filter width, f h = 0.016 Hz and f h = 0.0016 , respectively. h2 = 2 x 10-24 More convergent variances are introduced in F below and in Section VIII. F. Modified Allan Variance The relatively poor discrimination of cy(.) against white and flicker phase noise prompted the development of the modified Allan variance, mod c ~ ( T ) ,as shown below, in 1981 [30], [31]. G. Comments on mod mod c:(nTo) - ?42(7) . . . Y 2 n ( 7 ) - Yn(.,,) (12) Here the xi's are the time variations measured at intervals t o and ti(.) = -?Ci)/(n~o). See Fig. 6. Ti is the 956 phase averaged over n adjacent measurements of duration TO.Thus mod C: ( T ) is proportional to the second difference of the phase averaged over a time nT0. Viewed from the frequency domain, mod (T;(T) is proportional to the first difference of the frequency averaged over n adjacent ) equal to mod e $(.). samples. If n = 1 then ( T ~ ( T is This measurement process results in an equivalent noise bandwidth of f h l n when 2nfnro >> 1. Figure 7 shows the dependence of mod aY(n70) on measurement time nT0 for the five common noise types, in the limit that 2TfhTO >> 1 [26]-[28], [31], [34]. Figure 8 shows the ratio of mod e;(nTO)/c,2(nq,)versus n. U,(.) Mod e y ( n T O ) behaves very similarly to ey(nTo) for a = 0,-1,-2 and -3. Noise types a = 1 and a = 2 are easily separated using mod cy(n70). this approach is actually a software realization of the variable noise bandwidth proposal of [32]. In the presence of noise types a 2 1, mod e ( n T 0 ) depends on TO. By way of illustration, assume a = 2; then mod a y ( l O n ~ , ~=o ) 1/10-1/2 mod cy(ni,1070). It is therefore necessary to specify f h , nT0 and n or TO for measurements of PROCEEDINGS OF THE IEEE, VOL. 79, NO. 7, JULY 1991 S,(f)=h,fa 2 1 0.01 1 ,\" , , , , ,,,,, 2 5 1 0 2 , , ,, 5 1 0 0 2 5 ,,,I 1 m n, Number of Samples Averaged Fig. 8. Ratio of mod a;( T)' to a, ( T)' as a function of n, the number of points averaged to obtain mod ay(.) in the limit that 2 7 r f h ~ o is much larger than 1. The measurement time T = n.0, where TO is the minimum data interval. mod o , ( ~ T o ) whereas only f h and TO are required for a,(nq). The effect of noise types (Y = 1 and 2 on the determination of average frequency or time in practical sources can be eliminated by making n large enough or equivalently, TO small enough. Under this condition, mod a,(n~o)becomes independent of n for n greater than some minimum value which depends on the ratio of a = 1 and 2 noise to (Y = -1, - 2 and -3 noise. This extremely important result, first expounded by Bernier [34] in 1987, is now used to improve the determination of time interval and/or frequency in a number of precision applications. VII. CALCULATION OF U:(.) AND mod U ; ( T ) FROM THE FOURIERFREQUENCY DOMAIN Cutler and Searle derived the calculation of ay(.) from S,(f) [41: More generally, any variance is related to the spectral density S, (f) by an integral mathematical relationship (Section VIII, (20)) wherein the transfer function H ( f ) is a Fourier transform of a stepwise function associated with the measurement response in the time domain [3]. It can be shown, and this is the key point, that the general shape, degree of selectivity, and the behavior at very low Fourier frequencies of the transfer function give a good understanding of the specific properties of the related variance. The transfer function approach has also been used to develop new types of experimental measurement systems as will be shown in Section VIII. Much of this development has been made possible by recent advances in high speed digital processing and microprocessors. A . Application to Sinusoidal FM: Even the best sources are frequency modulated by unwanted sinusoidal signals. Although the above stability measures were developed to deal with random processes, sinusoidal instabilities do have an influence on them. 1) Fourier Frequency Domain: If AVO y ( t ) = -sin(% fmt) VO then the spectral density S,(f) contains a discrete line at the modulating frequency f,: where 6 is the Dirac delta function. Looking for discrete lines in spectral densities provides a convenient and powerful means to identify periodic variations. Their presence usually does not interfere with the identification of the slopes due to random noise. The substitution of (16) in (13) yields Substitution .of (16) into (14) yields Mod ay(.) can be calculated from the Fourier frequency domain using [21], [26]-[28], [31], [33] (14) The integrals in (13) and (14) can be calculated analytically for a number of simple cases [26], [31]; however, the general case is most easily evaluated numerically [27], [28]. For both integrals it is necessary to specify the value and shape of the low-pass filter for noise types with CY > 0. The most common shapes are the infinitely sharp low-pass filter with cut off frequency f h , and a single pole filter of equivalent noise bandwidth f h . The results for a = 2 are particularly dependent on the shape of the low-pass filter. See Figs. 4-9. Thus the effect of sinusoidal FM in both cases is 0 when T equals the modulation period T, = f;' or one of its multiples, since the modulating signal is completely averaged away. The largest value of mod o,(T) due to sinusoidial FM occurs when T is near T,/2 or one of its odd multiples [9]. Mod o , ( ~ T o ) falls n times faster than a,(n~o)for R f m n q >> R for sinusoidal FM. As a practical consequence, when caution has not been exercised about the relation between T, and the experimental values of 7, some scatter of the data results because of the oscillating behavior of (17) and (18). This scatter is added to the contribution due to the random noise(s) present as illustrated in Fig. 9. RUTMAN AND WALLS: FREQUENCY STABILITY IN PRECISION FREQUENCY SOURCES 957 m I I I modo ( 7 ) Y I I I 10’ 102 1 o3 H 1oo MEASUREMENT TIME (S) Fig. 9. Mod uy(.) versus 3 x 10-186(f - 0.125). T for the case of white phase noise (S,(f) = 4 x 10-’9f2, fh = 1, and frequency modulation with B. Application to Linear Frequency Drift: Equations (13) and (14) are not useful for evaluating c$(T) when linear frequency drift exists (i.e., y(t) = dit) since no tractable model seems to exist for Sy(f)in this case. Direct calculation in the time domain from (10) and (12) yields oY(7)= dl -T Jz di and mod oY(7)= - T . Thus, linear frequency drift yields a T and mod ay(t). Jz + law ~ for both ~ (19) ~ ( 7 VIII. OTHER MEASURESOF FREQUENCY STABILITY A number of other measures have been proposed and used during the past 20 years. Each one possesses some advantages and limitations compared to the well-established ones. The Hadamard variance [13] has been developed for making high resolution spectral analysis of y(t) from measurements of gk;that is, the frequency domain parameter Sy(f)is estimated from data provided by digital counters. The high-pass variance [14] has been developed through the “transfer function approach” wherein a variance is defined by the transfer function H ( f ) appearing in the general relationship (2~) It can be shown that an estimate of the Allan variance is provided by high-pass filtering the demodulated phase noise, without the need for counting techniques. A bandpass 958 sy(f)= variance has also been introduced [ 141 to solve the problem of separating CY = 1 form CY = 2, and yields a slope of 7-l for cr = 1 and T - ~ for / ~ CY = 2. A “filtered Allan variance” has also been used to separate the various noise types [29]. A modified three-adjacent-sample variance denoted as Cy(.) has also been proposed [3] in the framework of a more general finite-time frequency control method [46]. The new parameter, whose experimental estimation by counting techniques is not very complicated, has the same general behavior as oy(T ) for power-law spectral densities, except that it converges for both a = -3 and CY = -4 ) noise types. ( T ) varies as T for CY = 2 and T ~ for / a = 4 noise types. Another point of view was proposed in [47] which may appear more useful than cy(.) for certain kinds of prediction problems. It relies on a two-sample variance (with nonadjacent samples) which is studied versus T for a fixed value of 7 (not versus T as in the other measures). The structure function approach [15], [16] plays a unifying role in the sense that most of the time-domain parameters appear as particular cases of the general structure function concept. This concept finds application in the determination of polynomial frequency drifts. A comparison of these and other approaches can be found in [3], [lo], [13]-[16], [33]-[44]. cy IX. CONCLUSION We have briefly described some of the developments and features of frequency stability measures, both in the Fourier frequency domain and in the time-domain, Spectral densities play a key role in the sense that all the timePROCEEDINGS OF THE IEEE, VOL. 79, NO. 7, JULY 1991 ~ domain measures may be deduced from them, whereas general inversion of the integral formulas is usually very difficult if not impossible. It has been shown that the concept of the transfer function allows one to understand clearly the advantages and limitations of each parameter. The primary focus has been the Allan or two sample variance and the modified Allan variance because they are by far the most commonly used. They provide useful well-behaved measures for all random noise types found in precision oscillators and equipment used in signal processing. They are, however, general purpose measures and other measures may be more useful in specific cases [3]. Complete mathematical descriptions of these measures can be found in the included references. The contribution of many individuals must be recognized together with the initiating and coordinating role of organizations of NASA, NBS (now NIST), IEEE, and CCIR. ACKNOWLEDGMENT The authors express their thanks to numerous colleagues for fruitful and stimulating discussions on this topic. They would especially like to thank D.W. Allan, C. Audoin, J. A. Bames, A. DeMarchi, J.-J. Gagnepain, J. Groslambert, P. Kartaschoff, B. Picinbono, J. DePrins, and J. Uebersfeld. REFERENCES [11 IEE-NASA Symposium on Short-Term Frequency Stability, Goddard Space Flight Center, Greenbelt, MD, Nov. 23-24, 1964 Document NASA SP-80. 121 SDecial Issue on Freauencv Stabilitv. Proc. IEEE. vol. 54. pp. 101-338, Feb. 1986. 131 J. Rutman, “Characterization of Dhase and freauencv instabilities in precision frequency sources; fifteen years 6f prdgress,” proc. IEEE, vol. 66, pp. 1048-1174, 1978. [4] L.S. Cutler and C. L. Searle, “Some aspects of the theory and measurement of frequency fluctuations in frequency standards,” Proc. IEEE, vol. 54, pp. 89-100, 1966. [5] D. W. Allan, “Statistics of atomic frequency standards,” in Proc. IEEE, vol. 54, pp. 221-230, Feb. 1966. [6] J.A. Barnes, A. R. Chi, L. S. Cutler, D. J. Healey, D. B. Leeson, T.E. McGunigal, J.A. Mullen, W. L. Smith, R. L. Sydnor, R. Vessot and G. M. R. Winkler, “Characterization of frequency stability,” IEEE Trans. Instrum. Meas., vol. IM-20, pp. 105- 120, May 1971; also found in [21]. 171 IEEE Standard Definition of Phvsical Ouantities for Fundamental and Time Metrology (IEEE Sth. 1136). [8] F. L. Walls and S. R. Stein, “Accurate measurements of spectral density of phase noise in devices,” in Proc. 3lstAnn. Frequency ControlSymp., Atlantic City, NJ, June 1-3, 1977, pp. 335-343. [9] D. W. Allan and J. A. Bames, “Properties of signal sources and measurement methods,” in Proc. 35th Ann. Frequency Control Symp., 1981, pp. 1-47; also found in [21]. [lo] J. Rutman, “Relations between spectral purity and frequency stability,” in Proc. 28th Ann. Frequency Control Symp., Atlantic City, NJ, May 29-31, 1974, pp. 160-165. [ l l ] P. Lesage and C. Audoin, “Characterization of frequency stability: Uncertainty due to the autocorrelation function of the frequency fluctuations,” IEEE Trans. Instrum. Meas., vol. IM22, pp. 157-161, June 1973. (See also the corrections published in Mar. 1974 and Sept. 1976); also found in [21]. [12] J.A. Barnes, “Tables of bias functions, B1 and Bz, for variances based on finite samples of processes with power law spectral densities,” NBS Tech Note 375, Jan. 1969; also found in [21]. [13] R. A. Baugh, “Frequency modulation analysis with the Hadamard variance, in Proc. 25th Ann. Frequency Control Symp., Atlantic City, NJ, Apr. 1971, pp. 222-225. [141 J. Rutman, “Characterization of frequency stability: a transfer function approach and its application to measurement via filter- __ L 1 - ., ing of phase noise,” IEEE Trans, Instrum. Meas., vol. IM-23, pp. 40-48, Mar. 1974. [15] W.C. Lindsey and C.M. Chie, “Theory of oscillator instability based upon structure functions,” Proc. IEEE, vol. 64, pp. 1652-1666, Dec. 1976. [16] W.C. Lindsey and C.M. Chie, “Structure function of oscillator fractional phase and frequency fluctuations,” IEEE Trans. Instrum. Meas., vol. IM-26, pp. 420-421, Dec. 1977. [17] J.E. Gray and D.W. Allan, “A method for estimating the frequency stability of an individual oscillator,” in Proc. 28thAnn. Frequency Control Symp., Atlantic City, NJ, May 29-31, 1974, pp. 243-246. [18] A. L. Lance, W. D. Seal, and F. Labaar, “Phase noise and AM noise measurements in the frequency domain,” Infrared and Millimeter Waves, vol. 11, 1984; also found in [21]. [19] F. L. Walls, S. R. Stein, J. E. Gray, and D. J. Glaze, “Design considerations in state-of-the-art signal processing and phase noise measurement system,” in Proc. 30th Ann. Frequency Control Symp., Atlantic City, NJ, June 1976, pp. 269-274. [20] D.W. Allan and H. Daams, “Picosecond time difference measurement system, in Proc. 29th Ann. Frequency Control Symp. Atlantic City, NJ, May 28-30, 1975, pp. 404-411. [21] D.B. Sullivan, D. W. Allan, D. A. Howe, and F. L. Walls, Eds., “Characterization of clocks and oscillators,” NIST Tech Note 1337, Mar. 1990. [22] G. Kramer, “Digitale bestimmung von schwankung-spektren,” PTB, Jahresbericht 1975, p. 157. [23] F. L. Walls, C. M. Felton, A. J. D. Clements, and T. D. Martin, “Accuracy model for phase noise measurements, in Proc. 21st Ann. Precise Time and Time Interval Planning Meeting, Redondo Beach, CA, Nov. 1990, pp. 295-310. [24] C.A. Greenhall, “A method for using a time interval counter to measure frequency stability,” in Proc. 41st Ann. Frequency Control Symp., 1987, pp. 126-135. [25] J. A. Barnes and D. W. Allan, “Variances based on data with dead time between the measurement,” NIST Tech Note 1318, 1990; also found in [21]. [26] P. Lesage and T. Ayi, “Characterization of frequency stability: analysis of the modified Allan variance and properties of its estimate,” IEEE Trans. Instrum. Meas., vol. IM-33, pp. 332-337, 1984; also found in [21]. [27] F.L. Walls, J. Gray, A.O’Gallagher, L. Sweet, and R. Sweet, “Time-domain frequency stability calculated from the frequency domain: an update,” in Proc. 4th European Frequency and Time Forum, Neuchatel, Switzerland, Mar. 1990, pp. 197-204. [28] F. L. Walls, J. Gary, A. O’Gallagher, R. Sweet, L. Sweet, “Time domain frequency stability calculated from the frequency domain description; use of the SISINT software package to calculate time domain frequency stability from the frequency domain”, NISTIR 89-3916. [29] J. Groslambert, J. J. Gagnepain, F. Vernotte, and F.L. Walls, “A new ‘filtered Allan variance’ and its application to the identification of phase and frequency noise sources,” in Proc. 43rdAnn. Frequency Control Symp., 1989, pp. 326-330. [30] J. J. Snyder, “An ultra high resolution frequency meter,” Appl. Opt., vol. 19, pp. 1223-1225, 1980. [31] D. W. Allan and J. A. Barnes, “A modified “Allan variance” with increased oscillator characterization ability,” in Proc. 35th Ann. Frequency Control Symp., 1981, pp. 470-475; also found in [21]. [32] R. Vessot, L. Mueller, and J. Vanier, “The specification of oscillator characteristics from measurements made in the frequency domain,” Proc. IEEE, vol. 54, pp. 199-207, Feb. 1966. [33] S. R. Stein, “Frequency and time-Their measurement and characterization,” in Precision Frequency Control, vol. 2, ch. 12, 1985. [34] L.G. Bernier, “Theoretical analysis of the modified Allan variance,” Proc. 41st Ann. Frequency Control Symp., 1987, pp. 116-121. [35] V.F. Kroupa and L. Sojdr, “Modified Allan variance: transfer functions and practical applications,” CPEM Dig. 1984. [36] P. Tremblay, “Etude de I’Effet du filliage du signal sur la caracterisation de la stabilite de frequence,” These, UniversitC Laval, P.Q., Canada, 1985. [37] C.C.I.R., XIV th Plenary Assembly, Kyoto 1978, Vol. VII, “Standard frequencies and time signals,” International Telecommunication Union, Geneva, Switzerland. [38] CCIR Recommendation 686 (1990), “Glossary,” in Vol. 7 (Stan- R U T M A N AND WALLS:FREQUENCY STABILITY IN PRECISION FREQUENCY SOURCES 959 dard Frequencies and Time Signals), available from: International Telecommunication Union, General Secretariat-Sales Section, Place des Nations, CH-1211, Geneva, Switzerland. D. B. Percival, “Prediction error analysis of atomic frequency standards,” in Proc. 31st Annu. Frequency Control Symp., Atlantic City, NJ, June 1977, pp. 319-326. W. C. Lindsay and C. M. Chie, “Identification of power-law type oscillator phase noise spectra from measurements,” IEEE Trans. Instrum. Meas. vol. IM-27, pp. 46-53, Mar. 1978. F. L. Walls, D. B. Percival, and W. R. Irelan, “Biases and variances of several FlT spectral estimators as a function of noise type and number of samples,” in Proc. 43rd Ann. Frequency Control Symp., 1989, p. 336; also found in [21]. S. Stein, D. Glaze, J. Levine, J. Gray, D. Hilliard, D. Howe, and L. Erb, ‘‘Performance of an automated high accuracy phase measurement system,” in Proc. 36th Ann. Frequency Control Symp., 1982, pp. 314-320. J. Rutman and J. Groslambert, “Characterization and measurement of Frequency stability: lbenty five years of progress,” in Proc. 4th Ann. European Frequency and Time Forum, 1990, pp. 145-148. J. Groslambert, D. Fest, M. Olivier, and J. J. Gagnepain, “Characterization of frequency fluctuations by crosscorrelations and by using three or more oscillators,” in Proc. 35th Ann. Frequency Control Symp., 1981, pp. 458-463. J. J. Gagneapain, J. Groslambert, and R. Brendel, “The fractal dimension of phase and frequency noises: another approach to oscillator characterization,” Proc. 39th Ann. Frequency Control Symp., 1985, pp. 113-118. E. Boileau and B. Picinbono, “Statistical study of phase fluctuations and oscillator stability,” IEEE Trans. Instrum. Meas., vol. IM-25, pp. 66-75, 1976. J. De Prins and G. Comelissen, “Power spectrum, frequency stability and flicker noise,” in Proc. Frequency Standards and Metrology Seminar, Univ. Lava], P.Q., Canada, Aug. 30-Sept. 1, 1971, pp. 368-387. F. L.Walls was born in Portland, OR, on October 29, 1940. He received the B.S., M.S., and Ph.D. degrees in physics from the University of Washington, Seattle, in 1962, 1964, and 1970, respectively. His Ph.D. dissertation was on the development of long-term storage and nondestructive detection techniques for electrons stored in Penning traps and the first measurements of the anomalous magnetic (g-2) moment of low energy electrons. From 1970 to 1973 he was a Post-Doctoral Fellow at the Joint Institute for Laboratory Astrophysics in Boulder, CO. This work focused on developing techniques for long-term storage and nondestructive detection of fragile atomic ions stored in Penning traps for low energy collision studies. Since 1972 he has been a Staff Member of the Time and Frequency Division of the National Institute of Standards and Technology, formerly the National Bureau of Standards, in Boulder. He is presently engaged in research and development of hydrogen maser devices and cesium beam standards for use as ultra-stable clocks, crystal-controlled oscillators with improved short- and long-term stability, low-noise microwave oscillators, frequency synthesis from RF to infrared, low-noise frequency stability measurement systems, and accurate phase noise metrology. He has published more than 70 scientific papers and holds 3 patents. Dr. Walls is a member of the American Physical Society. Jacques Rutman (Senior Member, IEEE), was born in Paris, France, on November 24,1945. He received the Docteur de BBme Cycle degree in electronics and the Docteur 6s Sciences degree in physics from the University Paris VI, Paris, France in 1970 and 1972, respectively. From 1968 to 1971 he prepared his Thesis at the French Office National d’etudes et de Recherches Atrospatiales. From 1971 to 1974 he was with “Adret Electronique as Head of the Research Department. In 1975, he joined the Bureau National de Metrologie as Director of the Laboratoire Primaire du Temps et des FrCquences located at the Pans Observatory. From 1984 to 1986, he was with Framatome-Novatome as Deputy Director of their Research and Test Laboratories. In 1986, be joined CEPE, a subsidiary of Thomson-CSF, as technical Director. Since 1988, he is Deputy Technical Director of the FCdCration des Industries Electriques et Electroniques. He has been a member of Study Group 7 on Standard Frequencies and Time Signals of the CCIR from 1976 to 1984. He is a member of the ComitC National FranCais de RadioelectricitC Scientifique (CNFRS), the French Committee of the International Union of Radio Science, (URSI), since 1977. Dr. Rutman is a member of the French “SociCtC des Electriciens et des Electroniciens” (SEE) where he has been President of the Technical Section on Metrology, Measurement and Instrumentation (1977-1981) and President of the French Review L’Onde Electrique (1981-1987). Since 1988, he is also a member of the SociCtt des IngCnieurs et Scientifiques de France (ISF), and technical adviser to the French Review Electronique Europe 2000. 960 PROCEEDINGS OF THE IEEE, VOL. 79, NO. 7, JULY 1991 __ -
https://openalex.org/W3199557079	http://publikationen.ub.uni-frankfurt.de/files/64561/s41398-021-01598-y.pdf	English	null	Individuals at increased risk for development of bipolar disorder display structural alterations similar to people with manifest disease	Translational psychiatry	2,021	cc-by	9,460	1Department of Psychiatry and Psychotherapy, Carl Gustav Carus University Hospital, Technische Universität Dresden, Dresden, Germany. 2Core-Facility Brainimaging, Faculty of Medicine, University of Marburg, Marburg, Germany. 3Department of Psychiatry, University of Marburg, Marburg, Germany. 4Center for Mind, Brain and Behavior (CMBB), University of Marburg and Justus Liebig University Giessen, Marburg, Germany. 5Neuroimaging Center, Technische Universität Dresden, Dresden, Germany. 6Institute for Medical Informatics and Biometry, Carl Gustav Carus Faculty of Medicine, Technische Universität Dresden, Dresden, Germany. 7Department of Psychiatry and Psychotherapy, Tübingen Center for Mental Health, University of Tübingen, Tübingen, Germany. 8Department for Biomedical Resonance, University of Tübingen, Tübingen, Germany. 9Department of Psychiatry and Psychotherapy, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. 10Department of Psychiatry, Psychotherapy and Psychosomatic Medicine, Vivantes Hospital Am Urban and Vivantes Hospital Im Friedrichshain, Charité-Universitätsmedizin Berlin, Berlin, Germany. 11Department of Psychiatry, Psychosomatic Medicine and Psychotherapy, University Hospital, Goethe University, Frankfurt, Germany. 12Department of Psychiatry and Neurosciences, Charité Campus Mitte, Charité - Universitätsmedizin Berlin, Berlin, Germany. 13Department of Clinical Psychiatry and Psychotherapy, Brandenburg Medical School Theodor Fontane, Neuruppin, Germany. 14Department of Psychiatry, Psychotherapy and Preventive Medicine, LWL University Hospital, Ruhr-University, Bochum, Germany. 15Department of Child and Adolescent Psychiatry, Charité Universitätsmedizin Berlin, Berlin, Germany. 16Department of Psychiatry, Northwell Health, The Zucker Hillside Hospital, Glen Oaks, NY, USA. 17Department of Psychiatry and Molecular Medicine, Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, USA. ✉email: pavol.mikolas@uniklinikum-dresden.de ARTICLE OPEN Individuals at increased risk for development of bipolar disorder display structural alterations similar to people with manifest disease Translational Psychiatry (2021) 11:485 ; https://doi.org/10.1038/s41398-021-01598-y Translational Psychiatry Translational Psychiatry www.nature.com/tp Received: 1 July 2021 Revised: 6 August 2021 Accepted: 25 August 2021 ARTICLE OPEN Individuals at increased risk for development of bipolar disorder display structural alterations similar to people with manifest disease Pavol Mikolas 1✉, Kyra Bröckel1, Christoph Vogelbacher2,3,4, Dirk K. Müller1,5,6, Michael Marxen 1,5, Christina Berndt1, Cathrin Sauer1, Stine Jung1, Juliane Hilde Fröhner 1,5, Andreas J. Fallgatter7, Thomas Ethofer7,8, Anne Rau7, Tilo Kircher2,3,4, Irina Falkenberg 2,3,4, Martin Lambert9, Vivien Kraft 9, Karolina Leopold10, Andreas Bechdolf10, Andreas Reif 11, Silke Matura11, Thomas Stamm12,13, Felix Bermpohl12, Jana Fiebig12, Georg Juckel14, Vera Flasbeck 14, Christoph U. Correll15,16,17, Philipp Ritter 1, Michael Bauer 1, Andreas Jansen2,3,4 and Andrea Pfennig1 Pavol Mikolas 1✉, Kyra Bröckel1, Christoph Vogelbacher2,3,4, Dirk K. Müller1,5,6, Michael Marxen 1,5, Christina Berndt1, Cathrin Sauer1, Stine Jung1, Juliane Hilde Fröhner 1,5, Andreas J. Fallgatter7, Thomas Ethofer7,8, Anne Rau7, Tilo Kircher2,3,4, Irina Falkenberg 2,3,4, Martin Lambert9, Vivien Kraft 9, Karolina Leopold10, Andreas Bechdolf10, Andreas Reif 11, Silke Matura11, Thomas Stamm12,13, Felix Bermpohl12, Jana Fiebig12, Georg Juckel14, Vera Flasbeck 14, Christoph U. Correll15,16,17, Philipp Ritter 1 Michael Bauer 1, Andreas Jansen2,3,4 and Andrea Pfennig1 © The Author(s) 2021 © The Author(s) 2021 In psychiatry, there has been a growing focus on identifying at-risk populations. For schizophrenia, these efforts have led to the development of early recognition and intervention measures. Despite a similar disease burden, the populations at risk of bipolar disorder have not been sufﬁciently characterized. Within the BipoLife consortium, we used magnetic resonance imaging (MRI) data from a multicenter study to assess structural gray matter alterations in N = 263 help-seeking individuals from seven study sites. We deﬁned the risk using the EPIbipolar assessment tool as no-risk, low-risk, and high-risk and used a region-of-interest approach (ROI) based on the results of two large-scale multicenter studies of bipolar disorder by the ENIGMA working group. We detected signiﬁcant differences in the thickness of the left pars opercularis (Cohen’s d = 0.47, p = 0.024) between groups. The cortex was signiﬁcantly thinner in high-risk individuals compared to those in the no-risk group (p = 0.011). We detected no differences in the hippocampal volume. Exploratory analyses revealed no signiﬁcant differences in other cortical or subcortical regions. The thinner cortex in help-seeking individuals at risk of bipolar disorder is in line with previous ﬁndings in patients with the established disorder and corresponds to the region of the highest effect size in the ENIGMA study of cortical alterations. Structural alterations in prefrontal cortex might be a trait marker of bipolar risk. This is the largest structural MRI study of help-seeking individuals at increased risk of bipolar disorder. MATERIALS AND METHODS p In this study, we analyzed the MRI and baseline clinical data acquired in a subsample of the ongoing Early-BipoLife study. Details of the study protocol can be found elsewhere [25, 26]. Brieﬂy, the Early-BipoLife study is a multicenter, naturalistic, prospective-longitudinal observational cohort study performed at ten German universities and teaching hospitals with early detection centers/facilities and specialized in- and outpatient care. Although Early-BipoLife is an ongoing study, the baseline acquisition of clinical and MRI data has been completed. Of the total N = 1 229 recruited adolescents and young adults (age 15−35) at risk, N = 313 volunteered to receive an MRI (recruited at seven of the ten study sites: Berlin, Bochum, Frankfurt, Hamburg, Dresden, Marburg, Tübingen) [27]. In order to include all proposed risk factors for bipolar disorder, we recruited the participants in three recruitment pathways: N = 123 were consulting early detection centers/facilities and were screened positive for an indication of ≥1 proposed risk factor for bipolar disorder, N = 146 were young in- and outpatients with a depressive syndrome and N = 44 had an established diagnosis of ADHD. For the complete inclusion criteria for each recruitment pathway see Supplementary Note 2. Participants were phenotyped in depth including the above-mentioned instruments to assess the risk for developing bipolar disorder. For the demographic characteristics of the sample, see Table 1 and Supplementary Tables 2, 3. Although a positive family history was shown to be the most robust risk factor for bipolar disorder, only a fraction of individuals with ﬁrst-degree relatives will develop bipolar disorder (4.5 −22.4%) [18–20], some may actually develop other disorders, such as major depression. Additionally, most bipolar patients do not have a reliable positive family history. Beyond the genetic risk, several non-genetic risk factors were suggested to be associated with an increased risk for bipolar disorder. These include speciﬁc subsyndromal manic symptoms, depressive syndromes, mood swings, changes in sleep and circadian rhythm, anxiety disorders, attention deﬁcit hyperactivity disorder (ADHD), speciﬁc character traits, stressful life events, or substance abuse [5, 21]. Based on studies of these risk factors, several risk-assessment tools have been developed: Bipolar-at-risk (BAR) criteria [22], Extended BAR criteria (BARS) [23], Bipolar Prodrome Symptom Interview and Scale prospective version (BPSS-P) [24], Early Phase Inventory for Bipolar Disorders (EPIbipolar) [21] (see also Supplementary Table 1). INTRODUCTION I h associated with a disease burden comparable to psychotic disorders [4], the populations at risk have not been sufﬁciently identiﬁed and characterized [5]. In recent years, there has been an increasing effort to deﬁne and characterize individuals at risk for psychiatric disorders. These efforts gave rise to specialized early recognition services, which provide risk-stratiﬁcations and targeted interventions for help- seeking individuals at risk [1, 2]. As an example, cumulative evidence from epidemiological, genetic, neuroimaging, and interventional studies led to the establishment of the psychosis risk syndrome, which is now included as a diagnostic category in the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) [3]. Although bipolar disorder remains similarly prevalent and is Bipolar disorder has been associated with brain structural alterations. Whereas earlier structural magnetic resonance imaging (MRI) studies focused on cortical volume, more speciﬁc segmenta- tion methods allowed for its discrete and developmentally distinct quotients—cortical thickness and surface area [6–8]. Up to date, the ENIGMA group performed two large-scale multicenter studies, which analyzed structural differences between individuals with bipolar disorder and healthy participants [9, 10], thereby P. Mikolas et al. 2 bipolar disorder and healthy controls, respectively, and published the lists of effect sizes for particular regions. From those data, we selected one cortical and one subcortical ROI based on the largest effect size in the given study. More speciﬁcally, we selected the left inferior frontal gyrus—pars opercularis (a region within the broader ventrolateral prefrontal cortex (VLPFC)) from Hibar et al. [9] and hippocampus from Hibar et al. [10]. As Hibar et al. [9] identiﬁed differences only in cortical thickness and not in the surface area, we analyzed the differences in cortical thickness between the bipolar risk groups for pars opercularis. As the above- mentioned modalities do not apply for subcortical structures, and in accordance with Hibar et al. [10]. We analyzed the differences in gray matter volume in the case of hippocampus. Lastly, we also aimed to detect possible widespread differences in cortical thickness or subcortical volumes in exploratory analyses outside the pre-speciﬁed ROIs. To our knowledge, the present study is the largest structural MRI study of help-seeking individuals at increased risk of bipolar disorder. overcoming typical limitations of single studies or traditional meta- analyses, such as low power, publication bias, or comparability of results [11]. In the ﬁrst study, Hibar et al. [10]. INTRODUCTION I h investigated subcortical structures in 4304 participants and found reduced volumes of hippocampus, amygdala, and thalamus among individuals with bipolar disorder. In the second large-scale study, the authors [9] investigated cortical structures in a sample of 6503 participants and identiﬁed a pattern of signiﬁcant reductions of cortical thickness in frontal, temporal, and parietal regions, with maximum effect size in the left inferior frontal gyrus—pars opercularis (see below). Moreover, the reductions of cortical thickness correlated with illness duration. This study detected no differences in the surface area. According to a recent meta-analysis, volumetric studies in patients in early stages of illness consistently reported reduced volume of the bilateral pregenual anterior cingulate cortex [12]. Structural MRI studies in people at risk for bipolar disorder have so far exclusively focused on genetic risk, i.e. studying the affected or unaffected ﬁrst-degree relatives of bipolar patients. Interest- ingly, in contrast to patients with established disease, individuals with genetic risk seem to display rather increased cortical volume, particularly in the right inferior frontal gyrus, lingual gyrus, and superior temporal gyrus [13–15]. Higher cortical volume of the inferior frontal gyrus was proposed as a trait for genetic risk and may diminish as a result of disease progression or other contributing risk factors [14–16]. Regarding subcortical structures, there has been meta-analytical evidence for reduced amygdala volumes in adults with genetic risk while detecting no signiﬁcant effects in other subcortical structures including hippocampus, striatum, and thalamus [13, 17]. MATERIALS AND METHODS Exclusion criteria for study enrollment were as follows: diagnosis of bipolar disorder, schizoaffective disorder, schizophrenia; diagnosis of anxiety, obsessive-compulsive, or substance dependence disorder that fully explained the whole symptomatology; limited ability to comprehend the study; implied expressed negative declaration of intent to participate in the study by a minor; acute suicidality. The study was approved by the Ethics Committee of the Medical Faculty of the Technische Universität Dresden (No: EK290082014), as well as local ethics committees at each study site. We obtained a written informed consent after comprehensive information about study aims and procedures. In contrast with traditional studies of ﬁrst-degree relatives, in this study, we investigated structural alterations of the brain in help-seeking individuals at increased risk of bipolar disorders. This approach goes beyond the genetic risk approach to search for neural correlates of genetic, as well as non-genetic risk for bipolar disorder. We focused on the cortical thickness for the following reasons: 1. High-grade evidence for reduced cortical thickness in bipolar disorders in a large-scale multicenter study [9], 2. We hypothesized that the neural alterations in help-seeking indivi- duals may represent a diverse pattern of factors beyond genetics which may be associated with structural alterations rather similar to those in patients with established disease (i.e. reduced cortical thickness). Translational Psychiatry (2021) 11:485 Assessment of bipolar risk We used the EPIbipolar interview for the primary analyses presented here. EPIbipolar integrates the broadest range of early and late risk factors for bipolar disorder [21]. EPIbipolar is a semi-structured interview, which classiﬁes the participants according to the presence of main and secondary risk factors into no-risk, low-risk, and high-risk groups (for an overview see Supplementary Table 4). In this study, we used a modiﬁcation of the original risk categories according to Leopold et al. [21], which accounts for the low sensitivity of the original high-risk category. In this analysis, the original high-risk and ultra-high risk groups were fused, as the high-risk group contained a disproportionally low number of participants (3.2%). EPIbipolar was primarily designed as a risk stratiﬁcation tool for the purposes of targeted clinical intervention within early recognition services. From the clinical perspective, individuals with up to one secondary risk factor, or more secondary risk factors in a bipolar non-speciﬁc constellation might not beneﬁt from a targeted intervention [21]. For this reason, these participants were assigned to the ‘no-risk’ category. The EPIbipolar In order to perform an analysis of cortical and subcortical structural alterations and simultaneously attain a sufﬁcient statistical power, we performed a ROI approach (for details on the power calculation see Supplementary note 1). We based our choice on the two above-mentioned large-scale ENIGMA multi- center studies available at the time [9, 10]. Those studies detected cortical and subcortical differences between individuals with Translational Psychiatry (2021) 11:485 P. Mikolas et al. 3 Table 1. Socio-demographic characteristics. In order to assess the risk-associated structural alterations in help-seeking individuals, we divided the participants into no-risk, low-risk, and high-risk groups using the EPIbipolar assessment tool. No-risk Low-risk High-risk Test Post hoca N (%) (Ntotal = 263) 32 (12.2) 130 (49.4) 101 (38.4) Demographic Sex female/male (%) 10/22 (31.3/68.8) 60/70 (46.2/53.8) 57/44 (56.4/43.6) χ2 = 6.642 p = 0.036* No-risk ≠high-risk; χ2 = 6.166 p = 0.039* Age (SD) 24.13 (3.08) 24.78 (4.55) 25.13 (4.42) F(2,102.393) = 1.028 p = 0.361 Education 1. No degree/attending school (%) 0 (0.0) 8 (6.3) 4 (4.0) p = 0.633b 2. Secondary school (%) 6 (18.8) 18 (14.1) 18 (18.0) 3. High school (%) 26 (81.3) 102 (79.7) 78 (78.0) Recruitment pathway 1. Early recognition (%) 15 (46.9) 48 (36.9) 50 (49.5) 2. Depression (%) 5 (15.6) 68 (52.3) 41 (40.6) 3. Assessment of bipolar risk ADHD (%) 12 (37.5) 14 (10.8) 10 (9.9) Psychiatric medication Yyes (%) 11 (34.4) 82 (63.6) 53 (53.0) χ2 = 9.432 p = 0.009 No-risk ≠low-risk; χ2 = 8.955 p = 0.009 1. Antidepressants (%) 5 (15.6) 67 (51.5) 40 (39.6) 2. Antipsychotics (%) 1 (3.1) 25 (19.2) 11 (10.9) 3. Mood stabilizers (%) 0 (0.0) 7 (5.4) 4 (4.0) 4. Anxiolytics & Sleep (%) 1 (3.1) 8 (6.2) 7 (6.9) 5. Psychostimulants (%) 5 (15.6) 6 (4.6) 10 (9.9) Substance use Smoking status 1. Never smoked (%) 18 (56.3) 63 (48.5) 39 (38.6) p = 0.079b 2. Current smoker (%) 9 (28.1) 59 (45.4) 52 (51.5) 3. Past smoker (%) 5 (15.6) 8 (6.2) 10 (9.9) Cannabis present 0. No use (%) 26 (81.3) 90 (69.2) 74 (73.3) p = 0.830b 1. <1×/month (%) 1 (3.1) 11 (8.5) 9 (8.9) 2. ~1×/month (%) 0 (0.0) 8 (6.2) 6 (5.9) 3. 2−9×/month (%) 2 (6.3) 8 (6.2) 6 (5.9) 4. ≥10×/month (%) 3 (9.4) 13 (10.0) 6 (5.9) Cannabis lifetime 0. No use (%) 15 (46.9) 48 (36.9) 42 (41.6) p = 0.942b 1. <1×/month (%) 8 (25.0) 28 (21.5) 19 (18.8) 2. ~1×/month (%) 0 (0.0) 6 (4.6) 5 (5.0) 3. 2−9×/month (%) 3 (9.4) 17 (13.1) 11 (10.9) 4. ≥10×/month (%) 6 (18.8) 31 (23.8) 22 (21.8) Genetic risk First-degree relatives of BD patients N (%) 0 (0.0) 3 (2.3) 17 (16.8) Second-degree relatives of BD patients N (%) 0 (0.0) 0 (0.0) 8 (7.9) In order to assess the risk-associated structural alterations in help-seeking individuals, we divided the participants into no-risk, low-risk, and high-risk groups using the EPIbipolar assessment tool. Abbreviations. BAR-Criteria Bipolar-at-Risk-Criteria; BD Bipolar Disorder. ap-values have been adjusted using Bonferroni Correction. bFisher−Freeman−Halton’s exact test was used for variables with ≥1 expected cell counts <5. p < 0.05; p < 0.01; *p < 0.001. Table 1. Socio-demographic characteristics. In order to assess the risk-associated structural alterations in help-seeking individuals, we divided the participants into no-risk, low-risk, and high-risk groups using the EPIbipolar assessment tool. In order to assess the risk-associated structural alterations in help-seeking individuals, we divided the participants into no-risk, low-risk, and high-risk groups using the EPIbipolar assessment tool. In order to assess the risk-associated structural alterations in help-seeking individuals, we divided the participants into no-risk, low-risk, and high-risk groups using the EPIbipolar assessment tool. p p ap-values have been adjusted using Bonferroni Correction. alues have been adjusted using Bonferroni Correction. her−Freeman−Halton’s exact test was used for variables with ≥1 expected cell counts <5. p < 0.05; p < 0.01; p < 0.001 AR-Criteria Bipolar-at-Risk-Criteria; BD Bipolar Disorder. In order to assess the risk-associated structural alterations in help-seeking individuals, we divided the participants into using the EPIbipolar assessment tool. ap-values have been adjusted using Bonferroni Correction. bFisher Freeman Halton’s exact test was used for variables with ≥1 expected cell counts <5 p < 0 05; p < 0 01; p < 0 001 g p Abbreviations. BAR-Criteria Bipolar-at-Risk-Criteria; BD Bipolar Disorder. ap-values have been adjusted using Bonferroni Correction. b e risk-associated structural alterations in help-seeking individuals, we divided the participants into no-risk, low-risk, and hig assessment tool. Assessment of bipolar risk b Translational Psychiatry (2021) 11:485 P. Mikolas et al. 4 interview is being further developed and currently undergoing a longitudinal validation within the BipoLife study [25, 26]. we used the bilateral mean hippocampal volume as a dependent variable, while controlling for all above mentioned covariates/factors, as well as for the total estimated intracranial volume. We assessed the homogeneity of variances between the groups using the Levene test (p = 0.146, p = 0.145 for pars opercularis and hippocampus respectively). We corrected the p- values for two comparisons (for two ROIs analyzed) using the false discovery rate (FDR) correction [35]. g p y As there might be an overlap between bipolar risk and psychosis risk syndromes, we screened for the presence of a possible psychosis risk syndrome using PQ-16 self-report questionnaire [28]. In all participants scoring 6 or above, we evaluated psychosis risk status using the Structured Interview for Psychosis Risk Syndromes (SIPS) [29]. We performed the following post hoc analyses: ﬁrst, we tested for a linear effect of the risk on the cortical thickness. More speciﬁcally, we conducted a multiple regression analysis with bipolar risk, sex, current psychiatric medication, smoking status, present cannabis use, and study site as independent variables, and thickness of the left parsopercularis as the dependent variable. The bipolar risk was established as ordinale values 0, 1, and 2 corresponding to the EPIbipolar risk groups no-risk, low-risk, and high-risk respectively. We then continued to generate the regression equation considering all signiﬁcant independent variables. To create the diagram showing the linear effect of EPIbipolar risk on the thickness of the left parsopercularis while holding the other independent variables constant, we inserted the sample means of the remaining signiﬁcant independent variables (relative frequencies for dummy variables) into the regression equation and plotted the resulting equation (Supplementary Fig. 1). Second, we performed a whole-brain exploratory analysis using the Freesurfer Qdec tool available in FreeSurfer 6.0. We used a general linear model (GLM) to estimate the differences in cortical thickness at each vertex of the surface between the EPIbipolar risk groups. We used age and sex as covariates. We corrected the analysis for multiple comparisons using the FDR correction (p < 0.05). Third, we performed an exploratory analysis of the bilateral mean volumes of other subcortical structures (thalamus, amygdala, nucleus accumbens, caudate, globus pallidus, putamen) using generalized linear models as described above for hippocampal volume. ROI based primary analyses of left pars opercularis and hippocampus We detected a signiﬁcant difference in the mean thickness of the left pars opercularis [F(2, 245) = 4.475, p = 0.024 (FDR-corrected)] between the bipolar-risk groups (Fig. 1 and Table 2) with medium effect size (Cohen’s d = 0.47). Post-hoc tests revealed a signiﬁcant difference between the no-risk and high-risk individuals (p = 0.012, FDR-corrected) (Fig. 1). The low-risk individuals displayed numerically thinner cortex of the left pars opercularis compared to the no-risk individuals as well, while showing a greater thickness than the high-risk individuals. However, both these pairwise comparisons were not statistically signiﬁcant (p = 0.08 and p = 0.106 respectively, FDR-corrected). Finally, we performed a standardized quality control of the cortical and subcortical segmentations and parcellations according to the established protocols of the ENIGMA working group (http://enigma.ini.usc.edu/ protocols/imaging-protocols). Brieﬂy, we visually inspected the segmented regions according to the internal and external surface methods and performed statistical outlier detection. The outliers were subjected for further visual inspection. We discarded those subjects, who did not pass the quality control or displayed major segmentation errors (N = 3). This number was rather low, potentially due to the preceding, thorough quality assessment using MRIQC (see above). p y We detected no signiﬁcant difference in the mean volume of hippocampus [F(2, 244) = 0.640, p = 0.528 (FDR-corrected)] between the bipolar-risk groups (Table 3). Assessment of bipolar risk The p-values were adjusted using FDR correction (p < 0.05). Finally, we repeated the primary ROI-based analyses of left pars opercularis and hippocampus using the risk deﬁned by BARS and BPSS-P criteria (for details on the risk criteria see Supplementary Table 1). In accordance with the previous study [22], we used a modiﬁed variable representing the presence or absence of any BARS/BPSS-P criterion respectively (NBAR+ = 186 (70.7%), NBPSS-P+ = 56 (21.3%)). MRI scanning f The BipoLife neuroimaging consortium involved seven study sites with different hardware and software conﬁgurations. At six sites, data were acquired at Siemens Magnetom MR scanners (Trio, Skyra, Prisma). One site had a Philips Achieva scanner. A detailed description of the scanning protocol (including details on participating study sites, MR scanners, speciﬁc hardware conﬁgurations) can be found in Vogelbacher et al. [27]. Pulse sequence parameters were standardized across all sites to the extent permitted by each platform. All subjects were assessed with a large neuroimaging battery, involving both high-resolution structural T1-weighted images and functional measurements (for an overview, see Ritter et al. [26]). In the present study, we focused on the analysis of the T1-weighted MRI data. A detailed description of the pulse sequence parameters of all sites is given in Vogelbacher et al. [27]. For quality assurance, the MRI images were analyzed using the MRIQC tool [30]. MRIQC can qualify structural and functional MR images and highlights the results in a human-readable report. This report contains several metrics including a movement plot and a plot of the background noise. For this dataset, a visual inspection by two authors was performed. The focus of this inspection was the general quality of the data. A set of 23 subjects was excluded from further analysis due to strong movement, ghosting, or fold-over artifacts. A detailed description of the MRI quality control protocol is described elsewhere [27]. MRI preprocessing We used the freely available FreeSurfer 6.0 software to perform the cortical and subcortical segmentations [6, 31]. To speed up the preprocessing with FreeSurfer, the computation was conducted in parallel and distributed using the NICePype [32]. For the cortical parcellations, we used the Desikan-Killiany atlas [31, 33], obtaining cortical thickness values for 68 (34 left and 34 right) cortical regions. For the segmentation of subcortical regions, we used the standard volumetric segmentation atlas available in Freesurfer [29], obtaining gray matter volumes of 14 subcortical structures (7 left and 7 right). Statistical analysis We performed the primary, ROI-based statistical analyses using extracted cortical thickness values of the left pars opercularis and hippocampal volume in IBM SPSS Statistics (version 27). After quality assessment and discarding participants with missing values for the risk scores, N = 263 subjects were available for the statistical analysis. In our primary analysis, we analyzed structural differences between the three EPIbipolar risk groups (no-risk, low-risk, and high-risk) in two á priori selected ROIs— left pars opercularis and hippocampus (deﬁned as bilateral mean hippocampal volume, see below) using generalized linear models. For the left pars opercularis, in accordance with prior ENIGMA study of cortical structures in bipolar disorder [9], we used cortical thickness as the dependent variable, while controlling for age, sex, current medication (yes/ no), smoking status (current/past smoker/never smoked), lifetime and present (<6 months) cannabis use (for the detailed characteristics of variables and their dimensions see Table 1). We used the site as a random effect. The effect size for the signiﬁcant result was calculated from partial eta squared according to Cohen [34]. For the hippocampus, in accordance with prior ENIGMA study of subcortical structures in bipolar disorder [10], DISCUSSION h d In this study of help-seeking individuals at risk of bipolar disorder, we detected a signiﬁcantly thinner left inferior frontal gyrus—pars opercularis in the high-risk individuals compared to the no-risk individuals. Moreover, there was a negative trend in the low-risk individuals. A post hoc analysis revealed an inverse linear effect of the risk group on the thickness of the left pars opercularis. There were no differences in the cortical thickness in the whole-brain exploratory analysis. We either did not detect any differences in the volume of the hippocampus and other subcortical regions. In accordance with the studies of established bipolar disorder, this study reports thinner cortex in help-seeking individuals at risk of bipolar disorder. A previous, large-scale multicenter study detected a pattern of reduced thickness of frontal, temporal, and parietal cortex [9]. The signiﬁcantly thinner cortex of the left pars opercularis in high-risk individuals in our study corresponds to the region of reduced thickness with maximum effect size in the patients of established disease in Hibar et al. [9]. Other multicenter studies detected a similar pattern of reduced cortical thickness in patients with schizophrenia, but not in other disorders, such as major depression, ADHD, obsessive-compulsive disorder, or autism [11, 36]. This suggests, that individuals at risk of bipolar disorder display structural alterations similar to the bipolar/ schizophrenia spectrum. Table 3. Results of the generalized linear models using the hippocampal volume as an independent variable. Measure F p-value Partial Eta squared EPIbipolar risk 0.640 0.528 0.005 Sex 9.821 0.002 0.039 Smoking status 0.677 0.509 0.006 Medication 1.335 0.249 0.005 Site 11.566 0.000 0.221 Age 0.947 0.332 0.004 Cannabis present 0.094 0.760 0.000 Cannabis lifetime 0.197 0.868 0.004 ICV 108.688 0.000 0.308 Structural and genetic similarities between bipolar disorders and schizophrenia are well known [11, 36, 37]. The symptoms of psychosis prodrome might also overlap with bipolar prodrome [38]. However, in our sample, we speciﬁcally recruited participants with bipolar risk factors, while manifest psychotic disorders were an exclusion criterion. Of all included participants, only six (2.3%) fulﬁlled the psychosis-risk status using the SIPS assessment tool. For this reason, we do not consider psychosis risk being a cofounder in our analysis. ondary regression analysis of left pars opercularis volumes (FDR-corrected): amygdala [F(2, 244) = 0.721, p = 0.487], caudate [F(2, 244) = 1.135, p = 0.485], putamen [F(2, 244) = 1.727, p = 0.36], nucleus accumbens [F(2, 244) = 4.439, p = 0.078], thalamus [F(2, 244) = 1.914, p = 0.36], pallidum [F(2, 246) = 0.875, p = 0.487]. volumes (FDR-corrected): amygdala [F(2, 244) = 0.721, p = 0.487], caudate [F(2, 244) = 1.135, p = 0.485], putamen [F(2, 244) = 1.727, p = 0.36], nucleus accumbens [F(2, 244) = 4.439, p = 0.078], thalamus [F(2, 244) = 1.914, p = 0.36], pallidum [F(2, 246) = 0.875, p = 0.487]. Table 2. Results of the generalized linear models using the thickness of the left pars opercularis as an independent variable. Table 2. Results of the generalized linear models using the thickness of the left pars opercularis as an independent variable. Measure F p-value Partial Eta squared EPIbipolar risk 4.475 0.012 0.035 Sex 1.417 0.235 0.006 Smoking status 0.336 0.715 0.003 Medication 2.171 0.142 0.009 Site 3.489 0.002 0.079 Age 9.152 0.003 0.036 Cannabis present 1.069 0.302 0.004 Cannabis lifetime 0.106 0.745 0.000 ondary regression analysis of left pars opercularis Secondary regression analysis of left pars opercularis A post hoc analysis using a multiple regression revealed a signiﬁcant regression equation (F(13, 249) = 3.502, p < 0.001, R2 = 0.16) with a linear effect of the risk score on the thickness of the pars opercularis (β = −0.037, p = 0.004) (Supplementary Fig. 1). Secondary ROI based analysis using BARS and BPSS-P criteria There were no signiﬁcant differences between the participants fulﬁlling vs. not fulﬁlling any BARS criterion in the thickness of pars opercularis [F(1, 247) = 1.681, p = 0.196] or hippocampal volume [F(1, 246) = 0.082, p = 0.775]. There were no signiﬁcant differences between the participants fulﬁlling vs. not fulﬁlling any BPSS-P criterion in the thickness of pars opercularis [F(1, 246) = 3.289, p = 0.071] or hippocampal volume [F(1, 245) = 0.710, p = 0.4]. Translational Psychiatry (2021) 11:485 P. Mikolas et al. 5 High-risk Low-risk No-risk Mean cortical thickness (mm) 2.85 2.80 2.75 2.70 A. B. Fig. 1 ROI-based analysis of cortical thickness by individuals at increased risk for development of bipolar disorder. A Left inferior frontal gyrus—pars opercularis as deﬁned by the Desikan−Killiany atlas. B Mean thickness of the left pars opercularis. The post hoc tests revealed a signiﬁcantly thinner cortex of the left pars opercularis between no-risk and high-risk groups. The low-risk group tends to have lower thickness than the no-risk group, while having a greater thickness than the high-risk group. However, these differences were not signiﬁcant. * denotes statistical signiﬁcance at p = 0.012 (FDR-corrected). Error bars represent the 95% conﬁdence intervals. High-risk Low-risk No-risk Mean cortical thickness (mm) 2.85 2.80 2.75 2.70 * B. A. B. A. Fig. 1 ROI-based analysis of cortical thickness by individuals at increased risk for development of bipolar disorder. A Left inferior frontal gyrus—pars opercularis as deﬁned by the Desikan−Killiany atlas. B Mean thickness of the left pars opercularis. The post hoc tests revealed a signiﬁcantly thinner cortex of the left pars opercularis between no-risk and high-risk groups. The low-risk group tends to have lower thickness than the no-risk group, while having a greater thickness than the high-risk group. However, these differences were not signiﬁcant. * denotes statistical signiﬁcance at p = 0.012 (FDR-corrected). Error bars represent the 95% conﬁdence intervals. The signiﬁcance test displays uncorrected p-values. Exploratory analyses However, in this case, only 21.3% of participants fulﬁlled the criterion for any risk state. We detected a negative linear relationship between risk category and cortical thickness of the left pars opercularis. This suggests, that the cortical thickness is associated with the total number of risk factors, i.e. the magnitude of the risk. Cortical thickness was shown to decrease with age in a linear manner in young individuals, while different genes may contribute to cortical change in different ages [8, 16]. This phenomenon might be locally accelerated in individuals at risk for bipolar disorders. Longitudinal, structural MRI, and genetic analyses are necessary to assess the dynamic changes in individuals at risk. In our statistical model, the effect size of the thinner cortex was moderate (as deﬁned by Cohen’s d of 0.47). This is higher than the effect sizes in other above-mentioned multicenter studies of patients with established disease (Cohen’s d ≤0.293) [9, 10]. Compared to the multicenter studies mentioned, here we present a sample of adolescents and young adults in the early stages of the potential development of bipolar disorder with a low cumulative dose of medication. Whereas those studies pooled datasets acquired within independent single studies, the recruit- ment and data acquisition in our study was performed in each center according to the same study protocol. These factors might have contributed to the effect size. As the expected effect sizes were small, we performed a literature-based, hypothesis-driven ROI analysis. There might be other affected regions as well (for example the widespread prefrontal or temporal cortices), how- ever, our sample would not have signiﬁcant power to detect weak effects in a whole-brain exploratory analysis. The mean age of individuals in our sample was higher than the typical onset of bipolar disorder (24.84 (SD = 4.4)), which might suggest, that we were looking at individuals with a degree of resilience. Approximately 70% of all individuals would develop bipolar disorder by the age of 21 [48, 49]. However, the time-to- diagnosis by bipolar disorder is typically long. Depending on the structure of psychiatric services, it might take 8.7−12.4 years from the appearance of the ﬁrst symptoms to establish the diagnosis [47–50]. Due to the predominance of depressive symptoms, as well as difﬁculties to recognize hypomania, the most typical false diagnosis is unipolar depression [24, 48, 49]. For this reason, we cannot make conclusions on resilience. Exploratory analyses Correcting for cannabis use might be misleading, as substance abuse is one of the risk factors in EPIbipolar. However, here the corresponding risk-deﬁning item is not equivalent, as it evaluates increasing, periodic substance abuse, which is only one aspect of possible patterns of cannabis use. This criterion was met only in a few (1.5%) subjects in our sample. On the other hand, chronic cannabis use was associated with patterns of cortical thickness reductions also in prefrontal regions, including left pars opercularis [40–42]. For that reason, correcting for cannabis use in this structural MRI study is still appropriate. A considerable amount of individuals in our sample had Axis I diagnoses other than bipolar disorder. We also intentionally included young patients with diagnosed depression and/or ADHD. As these have been associated with increased risk for bipolar disorders, pooling these participants is an appropriate way to study bipolar risk. This is also in line with previous MRI studies which analyzed affected participants with genetic risk, who met the criteria for mood disorders [15]. As a result of the population design of this study, the resulting risk groups were not matched according to the recruitment pathway, resulting in more participants with diagnosed depression in the low-risk and the high-risk group. On the other hand, there were no signiﬁcant reductions of cortical thickness in the VLPFC/inferior frontal gyrus in major depression according to the ENIGMA working group [11, 43]. Patients with ADHD displayed reduction of average cortical thickness [11, 44], however, in our sample there were no differences in the number of participants recruited over the ADHD recruitment pathway between the risk groups. Interestingly, in a secondary analysis, we did not detect differences in cortical thickness of pars opercularis or hippocampal volume between individuals meeting and not meeting any BARS criterion. However, in our sample, the BARS positive group consisted almost exclusively of participants who fulﬁlled the criterion for depressive symptoms. According to a recent multicenter study, there was no involvement of VLPFC alterations in major depression [43]. However, several studies described differences in hippocampal volume [45, 46]. On the other hand, these might be missing in young individuals without recurrent episodes, who are predomi- nantly represented in our sample [47]. Similarly, we did not detect any signiﬁcant structural differences using BPSS-P. Interestingly, the p-value for pars opercularis was rather small, which might indicate a statistical trend towards reduction. Exploratory analyses p y y The whole-brain exploratory analysis of cortical thickness showed no signiﬁcant clusters displaying differences between the risk groups (FDR-corrected). The exploratory analysis of the volumes of the subcortical structures showed no signiﬁcant differences in Our ﬁndings of the thinner cortex are in contrast with previous studies of individuals at risk for bipolar disorder, which focused exclusively on affected and/or non-affected ﬁrst-degree relatives Translational Psychiatry (2021) 11:485 P. Mikolas et al. 6 of bipolar patients. These identiﬁed possible structural biomarkers of genetic risk, such as increased volume of the right inferior frontal gyrus, superior temporal gyrus, or increased intracranial volume [13–16]. At the same time, signiﬁcantly more participants with genetic risk were assigned into the high-risk category in our sample. This might be counterintuitive, however, from the neuroimaging point of view, the early markers of a genetic risk for bipolar disorder seem to follow a trajectory towards reduction of volume/thickness, as the prodromal state or the disease progress. The above-mentioned ﬁndings of increased cortical volume are the likely biomarkers of genetic risk, especially before the manifestation of psychiatric symptoms. During the disease progression, other, non-genetic factors might override those early markers. The volume of the right inferior frontal gyrus was the largest in non-affected participants with genetic risk, and correlated inversely with illness duration in a combined group of affected participants with genetic risk and young subjects with bipolar disorder [15]. Interestingly, in a machine learning study using structural MRI, non-affected individuals with genetic risk were more accurately differentiated from healthy controls than individuals with genetic risk who already displayed clinical symptoms [39]. Finally, large-scale analysis suggests rather a widespread reduction of cortical thickness in manifest bipolar disorder [9, 11]. In order to study a comprehensive composition of risk factors beyond the genetic risk, the Early-BipoLife study included young help-seeking individuals with symptoms. For this reason, in our primary hypothesis, we expected structural alterations more typical for a disease progression, rather than for genetic risk. Although we did not test for differences in cortical volume, our ﬁnding of reduced cortical thickness in the left inferior frontal gyrus (pars opercularis) makes a potential ﬁnding of increased volume less likely. For this reason, we conclude, that we do not observe structural alterations directly associated with genetic risk in our analysis. education status, as the majority of individuals in all groups attended high-school. Translational Psychiatry (2021) 11:485 Exploratory analyses We also used other instruments (BARS, BPSS-P) with better validated psychometric properties; however, the validation studies lack longitudinal validation (BPSS-P) [24, 54], were performed in small samples (BAR), or lack replication (BARS) [22]. However, as the early recognition of bipolar disorder is an emerging ﬁeld, the risk assessment tools are being further developed [25] (Supplementary Table 1). In our secondary analysis, there was a discrepancy between the statistically signiﬁcant results among the risk assessment instruments. Future studies with more power are necessary, to evaluate the reproducibility of signiﬁcant structural ﬁndings among the various risk instruments. 8. Wierenga LM, Langen M, Oranje B, Durston S. Unique developmental trajectories of cortical thickness and surface area. NeuroImage. 2014;87:120–6. 9. Hibar DP, Westlye LT, Doan NT, Jahanshad N, Cheung JW, Ching C, et al. Cortical abnormalities in bipolar disorder: an MRI analysis of 6503 individuals from the ENIGMA Bipolar Disorder Working Group. Mol Psychiatry. 2018;23:932–42. 10. Hibar DP, Westlye LT, van Erp TG, Rasmussen J, Leonardo CD, Faskowitz J. et al. Subcortical volumetric abnormalities in bipolar disorder. Mol Psychiatry. 2016;21:1710–6. 11. Ching C, Hibar DP, Gurholt TP, Nunes A, Thomopoulos SI, Abé C, et al. What we learn about bipolar disorder from large‐scale neuroimaging: ﬁndings and future directions from the ENIGMA Bipolar Disorder Working Group. Hum Brain Mapp. 2020. https://doi.org/10.1002/hbm.25098. 12. Keramatian K, Chakrabarty T, Saraf G, Pinto JV, Yatham LN. Grey matter abnormalities in ﬁrst‐episode mania: a systematic review and meta‐analysis of voxel‐based morphometry studies. Bipolar Disord. 2020. https://doi.org/10.1111/ bdi.12995. 13. Cattarinussi G, Di Giorgio A, Wolf RC, Balestrieri M, Sambataro F. Neural signatures of the risk for bipolar disorder: a meta‐analysis of structural and functional neuroimaging studies. Bipolar Disord. 2019;21:215–27. As this was a naturalistic, population-based observational study, a control group was not included. As a result, most of the participants displayed at least minor symptoms. Analyzing population-based samples using severity subgroups is a reason- able approach, which may bring relevant information [55]. Including control groups in future studies is essential to determine the speciﬁcity of the ﬁndings. Although the generalized linear models approach is suitable for analyses of unequal groups, the resulting sample size of the no-risk group was relatively small for a neuroimaging study. 14. de Zwarte S, Brouwer RM, Agartz I, Alda M, Aleman A, Alpert KI, et al. The association between familial risk and brain abnormalities is disease speciﬁc: an ENIGMA-relatives study of schizophrenia and bipolar disorder. Exploratory analyses On the other hand, we can assume that some individuals with depression in our sample might have an unrecognized bipolar disorder. g g Inferior frontal gyrus has been implicated as an important functional hub in emotion regulation and cognitive control. Several studies detected aberrant functional activation and/or functional disconnection of this region. Task-based fMRI studies showed lower activation of the inferior frontal cortex, particularly in response to emotionally salient stimuli in youth at high risk of bipolar disorder [51]. Functional disconnection of the inferior frontal gyrus was detected in young individuals with bipolar disorder using resting-state fMRI [52]. From the functional point of Collecting data using the same study protocol for each center under close supervision enabled us to control for diverse factors, which might contribute to differences in cortical thickness. Beyond age, sex, and current medication, we also controlled for smoking status, past and present cannabis use. The cortical thickness reduction of pars opercularis stayed signiﬁcant even with correcting for these confounders. We did not correct for Translational Psychiatry (2021) 11:485 P. Mikolas et al. 7 view, an impaired inhibitory function of the inferior frontal cortex, may represent a trait marker of vulnerability to bipolar disorder [51, 53]. presenting with subthreshold bipolar symptoms: results from a prespeciﬁed interim analysis of a multicenter, randomized, controlled study. Bipolar Disord. 2020. https://doi.org/10.1111/bdi.12894. presenting with subthreshold bipolar symptoms: results from a prespeciﬁed interim analysis of a multicenter, randomized, controlled study. Bipolar Disord. 2020. https://doi.org/10.1111/bdi.12894. 3. Salazar de Pablo G, Catalan A, Fusar-Poli P. Clinical validity of DSM-5 attenuated psychosis syndrome: advances in diagnosis, prognosis, and treatment. JAMA Psychiatry. 2020;77:311–20. Functionally, cortical thickness reﬂects the volume of cells in a cortical column, the basic cortical functional unit [16]. Most of the anatomical connectivity takes place within cortical columns, rather than amid brain regions. Cortical thickness was also shown to be more dependent on local or intrinsic factors, rather than on the input from subcortical structures [8]. Our results, therefore, suggest, that the reduced cortical thickness in VLPFC/pars opercularis might be an early trait marker of risk for bipolar disorders, rather than a secondary effect of structural alterations in other, functionally connected (for example subcortical) structures. 4. Ferrari AJ, Stockings E, Khoo JP, Erskine HE, Degenhardt L, Vos T, et al. The prevalence and burden of bipolar disorder: ﬁndings from the Global Burden of Disease Study 2013. Bipolar Disord. 2016;18:440–50. 5. Exploratory analyses Biol Psychiatry. 2019;86:545–56. 15. Hajek T, Cullis J, Novak T, Kopecek M, Blagdon R, Propper L, et al. Brain structural signature of familial predisposition for bipolar disorder: replicable evidence for involvement of the right inferior frontal gyrus. Biol Psychiatry. 2013;73:144–52. 16. Drobinin V, Slaney C, Garnham J, Propper L, Uher R, Alda M, et al. Larger right inferior frontal gyrus volume and surface area in participants at genetic risk for bipolar disorders. Psychol Med. 2019;49:1308–15. In summary, we detected structural alterations similar to patients with manifest bipolar disorder, which correlate with the amount of known risk-factors. We detected reduced cortical thickness in a region, which has been functionally implicated as a potential biomarker of bipolar risk [51, 53]. Subcortical structures might not be structurally affected. Our secondary analyses, as well as large-scale multicenter studies of psychiatric disorders, do not suggest, that our ﬁnding of the thinner cortex was due to another diagnosis. 17. Fusar-Poli P, Howes O, Bechdolf A, Borgwardt S. Mapping vulnerability to bipolar disorder: a systematic review and meta-analysis of neuroimaging studies. J Psy- chiatry Neurosci. 2012;37:170–84. 18. Hafeman DM, Merranko J, Goldstein TR, Axelson D, Goldstein BI, Monk K, et al. Assessment of a person-level risk calculator to predict new-onset bipolar spec- trum disorder in youth at familial risk. JAMA Psychiatry. 2017;74:841–7. 19. Post RM, Altshuler LL, Kupka R, McElroy SL, Frye MA, Rowe M, et al. Multi- generational transmission of liability to psychiatric illness in offspring of parents with bipolar disorder. Bipolar Disord. 2018;20:432–40. g As this is an emerging ﬁeld, the concept of the risk for bipolar disorders is being further developed. We based the risk deﬁnition on the presence of known risk factors according to a systematic literature review. Longitudinal studies, as well as studies including healthy controls, are necessary, to evaluate the exact predictive validity and speciﬁcity of our ﬁndings. An interesting approach for future studies would be to include patients with psychosis risk, in order to identify speciﬁc structural differences between risk syndromes displaying possibly overlapping structural differences. Machine learning studies may provide a tool to extract the information from cortical thickness into individual risk- stratiﬁcation and/or risk-prediction tools [9, 56, 57]. 20. Kerner B. Genetics of bipolar disorder. Appl Clin Genet. 2014;7:33–42. 21. Leopold K, Ritter P, Correll CU, Marx C, Özgürdal S, Juckel G, et al. Exploratory analyses Faedda GL, Baldessarini RJ, Marangoni C, Bechdolf A, Berk M, Birmaher B, et al. An International Society of Bipolar Disorders task force report: precursors and pro- dromes of bipolar disorder. Bipolar Disord. 2019;21:720–40. p g y p disorders, rather than a secondary effect of structural alterations in other, functionally connected (for example subcortical) structures. Our work has several limitations. We used the risk for bipolar disorder according to the EPIbipolar interview for our analyses. We do not know yet, which participants will in the future develop bipolar disorder. However, the composition of risk factors giving rise to the risk categories was based on state-of-the-art evidence in the ﬁeld. We also used other instruments (BARS, BPSS-P) with better validated psychometric properties; however, the validation studies lack longitudinal validation (BPSS-P) [24, 54], were performed in small samples (BAR), or lack replication (BARS) [22]. However, as the early recognition of bipolar disorder is an emerging ﬁeld, the risk assessment tools are being further developed [25] (Supplementary Table 1). In our secondary analysis, there was a discrepancy between the statistically signiﬁcant results among the risk assessment instruments. Future studies with more power are necessary, to evaluate the reproducibility of signiﬁcant structural ﬁndings among the various risk instruments. As this was a naturalistic, population-based observational study, a control group was not included. As a result, most of the participants displayed at least minor symptoms. Analyzing population-based samples using severity subgroups is a reason- able approach, which may bring relevant information [55]. Including control groups in future studies is essential to determine the speciﬁcity of the ﬁndings. 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https://openalex.org/W2133416049	https://www2.ifrn.edu.br/ojs/index.php/HOLOS/article/download/1077/661	Portuguese	null	TRANSMISSÃO DO CONHECIMENTO DE ALGUMAS ZOONOSES PARA ALUNOS DE ESCOLAS PÚBLICAS NOS MUNICÍPIOS DE APODI, FELIPE GUERRA E SEVERIANO MELO	Holos	2,013	cc-by	7,470	RESUMO conhecimento da forma de transmissão da Larva migrans, Toxoplasmose e Leptospirose, onde antes das palestras o percentual era de 91,8, 1,9 e 29,4% e após as apresentações foi de 100,0, 57,8 e 72,4%, respectivamente. Para a forma de transmissão das Verminoses, o percentual de fixação do conhecimento não foi alterado, de maneira significativa, antes (86,5%) e após (87,9%) as palestras. Como conclusão, tem-se que a metodologia de palestras para alunos da rede pública de ensino é uma ferramenta que pode ser utilizada como forma de educação e prevenção de doenças. O presente trabalho objetivou avaliar o conhecimento de alunos da rede pública de ensino sobre algumas Zoonoses, através de palestras ministradas por alunos do curso de Técnico em Zootecnia. Foram aplicados 677 questionários para alunos de escolas públicas de Apodi, Felipe Guerra e Severiano Melo. Os questionários foram aplicados antes e após as palestras. Os dados foram discutidos através de uma análise descritiva. Os principais resultados demonstraram que os conteúdos relativos à transmissão e prevenção de algumas zoonoses foram fixados. Antes das palestras 20,8% dos alunos conheciam o conceito de Zoonoses e após, esse percentual passou para 88,3%. Houve um aumento no PALAVRAS-CHAVE: doenças, escolas, extensão, palestras, saúde pública. F. R. C. Moreira1, N. R. L. Morais1, F. L. M. Oliveira1, J. C. Souza1, M. S. Lima1, F. P. Costa1, P. V. S. Q. Moreira2, J. K. Góis1 ¹IFRN, campus Apodi; 2UERN, campus Mossoró faviano.moreira@ifrn.edu.br Artigo submetido em setembro/2012 e aceito em abril/2013 Artigo submetido em setembro/2012 e aceito em abril/2013 MOREIRA ET AL. (2013) AVALIAÇÃO DO CONHECIMENTO DE ALGUMAS ZOONOSES EM ALUNOS DE ESCOLAS PÚBLICAS NOS MUNICÍPIOS DE APODI, FELIPE GUERRA E SEVERIANO MELO (RN) - BRASIL F. R. C. Moreira1, N. R. L. Morais1, F. L. M. Oliveira1, J. C. Souza1, M. S. Lima1, F. P. Costa1, P. V. S. Q. Moreira2, J. K. Góis1 ¹IFRN, campus Apodi; 2UERN, campus Mossoró faviano.moreira@ifrn.edu.br INTRODUÇÃO As Zoonoses representam uma constante preocupação do poder público e da sociedade, uma vez que é frequente o contato entre animais e os seres humanos, seja para o trabalho, companhia ou fonte de alimentação. Em pequenas cidades, devido à ausência de uma estrutura efetiva de prevenção dessas doenças, as enfermidades transmitidas pelos animais aos seres humanos constituem um risco maior para a população. Nesse sentido, os trabalhos de educação em saúde possuem importância, principalmente para crianças e adolescentes. As zoonoses são consideradas um grande problema de saúde pública, pois representam 75% das doenças infecciosas emergentes no mundo. Estudos demonstram que 60% (849/1.415) dos patógenos humanos são zoonóticos e que 80% dos patógenos animais têm múltiplos hospedeiros (Ministério da Saúde, 2010). A transmissão das zoonoses para o homem geralmente ocorre diretamente pelo contato com animais infectados, os quais eliminam o agente pelas secreções; e indiretamente, pela ingestão de animais ou água contaminada. A participação de vetores e a interação com o ambiente também pode ocorrer. Por esta razão alimentos de origem animal, como carne, leite e derivados, ovos e mel são de grande importância (Langoni, 2004). Segundo Baltazar et al. (2004), os problemas relacionados à saúde animal e, consequentemente, à saúde pública podem ser minimizados quando se aplica a educação em saúde. Esses mesmos autores discorrem que para garantir uma relação homem-animal saudável é necessário educar as pessoas, não simplesmente com propagandas e divulgações em massa nos meios de comunicação, mas também com programas sistemáticos de educação em saúde, devidamente direcionados ao público alvo. Para Lima et al. (2010), uma das contribuições da promoção de saúde é a ampliação do seu entendimento, contribuindo para o processo em que a comunidade aumente a sua habilidade de resolver seus problemas de saúde com competência e intensifique sua própria participação. Segundo Silva Neto et al. (2002), levar informação para determinadas comunidades é um dos papeis fundamentais das instituições de ensino superior, melhorando as condições de vida da população e levando seus estudantes a colocar em prática os ensinamentos teóricos ouvidos em sala de aula, fortalecendo o binômio ensino – aprendizado. Para Oliveira et al. (2008), o foco da passagem do conhecimento dever ser em especial para crianças e jovens, por estes serem disseminadoras, repassando para os pais e outros adultos o aprendizado obtido. ABSTRACT this percentage increased to 88.3%. There was an increase in knowledge of transmission form of larva migrans, toxoplasmosis and leptospirosis, where before the presentation the percentage was 91.8, 1.9 and 29.4% and after was 100.0, 57.8 and 72.4%, respectively. For the transmission of intestinal worms, the percentage fixation of knowledge was not altered significantly before (86.5%) and after (87.9%) presentations. The conclusion is that the methodology of presentation to students in public education is a tool that can be used as a form of education and disease prevention. This study aimed to assess the knowledge of students from public schools on some Zoonosis, through presentation given by students of Animal Science Technician. 677 questionnaires were administered to public students in cities of Apodi, Felipe Guerra and Severiano Melo. The questionnaires were administered before and after. The data were discussed through a descriptive analysis. The main results showed that the content of transmission and prevention of some zoonosis are fixed. Before the presentation 20.8% of students did know the concept of Zoonosis and after, This study aimed to assess the knowledge of students from public schools on some Zoonosis, through presentation given by students of Animal Science Technician. 677 questionnaires were administered to public students in cities of Apodi, Felipe Guerra and Severiano Melo. The questionnaires were administered before and after. The data were discussed through a descriptive analysis. The main results showed that the content of transmission and prevention of some zoonosis are fixed. Before the presentation 20.8% of students did know the concept of Zoonosis and after, KEY-WORDS: diseases, extension, lectures, public health, schools KEY-WORDS: diseases, extension, lectures, public health, schools HOLOS, Ano 29, Vol. 2 66 MOREIRA ET AL. (2013) MOREIRA ET AL. (2013) HOLOS, Ano 29, Vol. 2 MATERIAL E MÉTODOS O trabalho foi desenvolvido pelos alunos do curso técnico em Zootecnia do IFRN campus Apodi em 12 escolas nos âmbitos municipal (E.M.), estadual (E.E.) e federal. Em Apodi foram atendidas as E.M. Lourdes Mota, E.M. Lindaura Silva, E.M. Francisco Targino da Costa, E.E. Professor Antonio Dantas, E.E. Professor Gerson Lopes, E.E. Zenilda Gama e IFRN. Em Felipe Guerra foram as E.M. José do Patrocínio Barra e E.E. Antonio Francisco e em Severiano Melo foram as E.M. Ricardo Sérgio, E.E. Severiano Melo e E.E. Américo Holanda. Foram aplicados 677 questionários e 1.922 alunos entre o 5º e o 9º ano do ensino fundamental e o 1º e o 3º ano do ensino médio nas modalidades integrado e ensino de jovens e adultos (EJA) assistiram as palestras. Os questionários foram elaborados com perguntas abertas e fechadas de acordo com Almeida Filho e Rouquayrol (2002), contendo informações sobre o conceito de zoonoses, relação entre roer unhas e verminose, relação entre andar descalço e verminose, transmissão de vermes entre animais e humanos, transmissão de doenças pelas fezes de gato, transmissão da leptospirose, presença de ratos nas residências dos alunos e forma de combate dos mesmos. Os questionários aplicados antes das palestras foram entregues aos alunos para serem respondidos durante as aulas, em espaço cedido pelos professores. Após as palestras, os questionários foram aplicados, como parâmetro para avaliar a assimilação do conteúdo e a qualidade da palestra realizada (Amaral et al., 2009). Em cada uma das escolas pesquisadas, foi definido um percentual de 10% de alunos que responderiam aos questionários. As perguntas foram as seguintes: As perguntas foram as seguintes:  Zoonoses são doenças transmitidas dos animais para os homens e dos homens para os animais?  Zoonoses são doenças transmitidas dos animais para os homens e dos homens para os animais?  Andar descalço influi quanto a adquirir verminose?  Roer unhas influencia quanto a adquirir verminose?  Os vermes dos animais são transmitidos para o homem?  As fezes do gato podem transmitir doenças? Se sim, qual?  Você sabe como se pega a Leptospirose?  A sua casa possui ratos? Se sim, Como os ratos são combatidos? Nas palestras foi utilizada uma linguagem simples e acessível, com recurso de multimídia – apresentação de slides com esquemas, ilustrações e conceitos dentro do tema proposto, onde as mesmas duravam, em média, 40 minutos. MOREIRA ET AL. (2013) nacional que foi de 10,0%. Para a raiva, no ciclo urbano, o RN é o 9º estado com maior número de notificações (Brasil, 2011). nacional que foi de 10,0%. Para a raiva, no ciclo urbano, o RN é o 9º estado com maior número de notificações (Brasil, 2011). Dessa forma, o objetivo do trabalho é avaliar a percepção dos alunos da rede pública de ensino sobre algumas zoonoses, antes e após a intervenção educativa na forma de palestras. MATERIAL E MÉTODOS Os temas das palestras foram: Toxoplasmose, Amebíase, Larva migrans e Leptospirose com ênfase nos conceitos de zoonoses e as linhas gerais do ciclo, transmissão, sinais clínicos das doenças e prevenção. A análise dos dados foi realizada de maneira descritiva e os mesmos apresentados em percentuais. INTRODUÇÃO Nesse sentido, a chegada do Instituto federal de Educação, Ciência e Tecnologia do Rio Grande do Norte (IFRN) na cidade de Apodi pode representar uma oportunidade de transferir informações úteis para a população, pois conforme Fensterseifer e Wiest (1986), tanto em zonas rurais como urbanas, os grupos comunitários e entre eles, as escolas, tem a maior importância no planejamento e na execução de programas de prevenção de zoonoses. As Zoonoses estão presentes no Rio Grande do Norte (RN) e em especial na região Oeste do estado, onde se localizam os municípios de Apodi, Felipe Guerra e Severiano Melo. Alguns exemplos podem ser observados no relatório de situação epidemiológica publicado pelo Ministério da Saúde (Brasil, 2011), onde 100% dos casos de Leishmaniose tegumentar e 47% de Leishmaniose visceral ocorreram no Oeste Potiguar. Quanto à leptospirose, no RN, em 2010 ocorreram 03 óbitos que representaram uma taxa de letalidade de 15,0%, maior que a média HOLOS, Ano 29, Vol. 2 67 RESULTADOS RESULTADOS HOLOS, Ano 29, Vol. 2 68 68 MOREIRA ET AL. (2013) A idade média dos 677 alunos que participaram do diagnóstico foi de 16,8 anos, onde os mesmos cursavam entre o 5º e o 9º ano do ensino fundamental e entre o 1º e o 3º ano do ensino médio, nas modalidades integrado e EJA. Para a tabela 1, pode ser observado o percentual de acertos e erros dos alunos frente ao conceito de zoonoses, onde antes das palestras 20,8% dos entrevistados souberam responder corretamente ao conceito de zoonoses e após as palestras, esse percentual passou para 88,3%. Tabela 1 - Conhecimento do significado de zoonoses por alunos da rede pública de ensino nos municípios de Apodi, Felipe Guerra e Severiano Melo no RN, antes e após as palestras serem ministradas (n=677) Momentos Zoonoses são doenças transmissíveis do ser humano para os animais e dos animais para o ser humano? Sim Não Não responderam Antes das palestras 20,8% 77,9% 1,3% Após as palestras 88,3% 11,4% 0,4% Tabela 1 - Conhecimento do significado de zoonoses por alunos da rede pública de en municípios de Apodi, Felipe Guerra e Severiano Melo no RN, antes e após as palestra ministradas (n=677) Na presente pesquisa, o percentual de respostas corretas a respeito do conceito de Zoonoses antes das palestras foi semelhante aos observados por Lima et al. (2010),que trabalharam com pais de alunos do pré-escolar da rede pública e particular de ensino em Recife- PE, esses autores encontraram que 28,21% (escola particular) e 28,0% (escola pública) dos pais definiram corretamente o termo Zoonoses. Esses dados mostram a necessidade de reforçar as noções de conceito das doenças transmitidas entre animais e seres humanos, fato observado no aumento das respostas corretas após as palestras. Almeida et al. (2009), trabalharam com crianças do 3º e 4º anos do ensino fundamental o conceito de zoonoses através de palestras e segundo os autores a maioria das crianças respondeu corretamente o conceito de zoonoses (68,5%), entretanto 26,3% responderam incorretamente e 5,2% deixaram em branco. Para os autores fatores como a falta de concentração e a quantidade de assuntos abordados durante a palestra podem ter confundido os alunos. Segundo Lima et al. (2010), muitas pessoas não conseguem distinguir ou não obtiveram a informação de forma correta de quais doenças os animais não adquirem, nem podem transmitir. Oliveira et al. MOREIRA ET AL. (2013) Tabela 2 - Conhecimento sobre o fato de andar descalço e a transmissão de verminoses em alunos da rede pública de ensino nos municípios de Apodi, Felipe Guerra e Severiano Melo no RN, antes e após as palestras (n=677) Momentos Andar descalço influi quanto a adquirir verminose? Sim Não Não responderam Antes das palestras 91,8% 7,9% 0,3% Após as palestras 100,0% 0,0% 0,0% 2 - Conhecimento sobre o fato de andar descalço e a transmissão de verminoses em a rede pública de ensino nos municípios de Apodi, Felipe Guerra e Severiano Melo no RN, antes e após as palestras (n=677) Tome et al. (2005) pesquisaram a percepção de professoras de ensino infantil sobre a relação entre andar descalço e verminose e foi constatado que 96,47% afirmaram que esse hábito influencia na aquisição de helmintoses. Segundo Bellato (2010), Larva migrans cutânea é um termo clínico que designa uma erupção dérmica de caráter linear e serpiginoso, produzida por larvas de alguns Nemathelminthes, normalmente parasitas do intestino delgado de cães e gatos, porém, podem atingir a pele do homem. Os caninos, como principais hospedeiros, propagam as parasitoses, através da contaminação ambiental por ovos e larvas de helmintos eliminados juntamente com as fezes. A doença é conhecida por dermatite serpiginosa, dermatite linear serpiginosa e bicho geográfico. A Larva migrans cutânea ocorre mais frequentemente em áreas tropicais e subtropicais e o problema é mais comum em pessoas que frequentam praias e terrenos arenosos, poluídos com fezes de cães e gatos. As crianças contaminam-se principalmente ao brincar em depósitos de areia para construções e em locais com areia destinados a recreação onde existe circulação de cães e gatos. As partes que mais frequentemente entram em contato com o solo são as mais sujeitas como pés, pernas, mãos e antebraços. Em crianças que brincam sentadas no chão, normalmente na região glútea e coxas, em frequentadores de praias as larvas podem penetrar em outras partes do corpo que normalmente ficam protegidas pela roupa (Bellato, 2010). Algumas medidas preventivas são: manter os animais em boas condições de higiene, sempre tratar os animais positivos, impedir o acesso de cães em locais frequentados por pessoas, em especial crianças, atuar em campanhas de conscientização, com orientações nas escolas e na comunidade (Bellato, 2010). RESULTADOS (2010) trabalharam com alunos ingressantes no curso de Biologia da UFRPE e verificaram que 55% dos alunos ingressantes afirmaram já ter ouvido falar sobre zoonoses, e os outros 45% nunca haviam escutado este termo. Na presente pesquisa, mesmo com um percentual de 88,3% de respostas positivas frente ao termo zoonoses, após as palestras, pode-se supor que uma taxa ainda maior não foi alcançada devido a fatores como: heterogeneidade dos alunos nas diversas escolas, com idades variando entre 11 e 60 anos; alguns colégios com salas superlotadas; salas com a acústica não apropriada ou; falta de concentração dos alunos. Na Tabela 2 são apresentados os dados referentes à relação entre andar descalço e a transmissão de doenças. Antes das palestras 91,8% sabiam dessa relação e após as mesmas, esse percentual subiu para 100%. Uma das principais doenças transmitidas ao andar descalço é a Larva migrans. HOLOS, Ano 29, Vol. 2 69 MOREIRA ET AL. (2013) Bezerra et al. (2003) ao trabalharem com crianças entre 1 e 6 anos em uma creche na cidade de Fortaleza, encontraram que existe correlação entre o tipo de parasita achado nas fezes e nas unhas indicando a provável participação dos depósitos sub-ungueais na transmissão dos parasitos. Tome et al. (2005) pesquisaram a percepção de professoras de ensino infantil sobre a relação entre roer unhas e verminose e foi constatado que 85,88% das educadoras afirmaram roer unhas influencia na aquisição de verminoses. Segundo Cordeiro e Macedo (2007), a amebíase que é causada pela Entamoeba histolyticae está presente em países de clima tropical onde a população é carente e não há saneamento básico. Segundo os mesmos autores, o homem se infecta ingerindo a forma cística madura contida em alimentos, água ou por qualquer tipo de contato fecal-oral. Também são possíveis formas menos usuais de transmissão, o sexo anal e oral e equipamentos de lavagem intestinal contaminados. Alguns sintomas da amebíase são disenteria, colite, apendicite, megacólon, peritonite, abscesso hepático, abscesso pleuropulmonar, lesões oculares e genitais. Por ser um parasito que causa milhares de mortes anualmente, é necessário que mais atenção seja dada a ele, principalmente pelas autoridades, já que o saneamento básico é a melhor forma de prevenir a doença (Cordeiro e Macedo, 2007). Segundo Costa et al. (2009), em função do desconhecimento dos princípios básicos de higiene, como o hábito de não lavar as mãos após evacuarem, muitas vezes levando-as à boca; com o uso de chupetas contaminadas e com o hábito de roer unhas, uma vez que elas podem abrigar uma série de ovos e cistos de parasitas, a incidência de amebíase aumenta, principalmente entre crianças. Na presente pesquisa, as respostas afirmativas frente à relação entre roer unhas e verminose diminuíram após as palestras, provavelmente pela forma de abordagem, onde o tema deveria ter sido abordado de maneira mais enfática. As escolas que tiveram o menor desempenho foram as que possuíam alunos com menor faixa etária (municipais). Dessa forma, pode ser que para estudantes com idade menor, a estratégia de transmissão do conhecimento não seja somente através de palestras e sim com o auxílio de outras metodologias como jogos educativos e oficinas. Os percentuais de conhecimento sobre a relação entre andar descalço e verminoses antes das palestras pode ser atribuído ao trabalho educativo que as professoras executam, uma vez que as noções básicas de higiene são trabalhadas desde as mais tenras idades dos estudantes. Todavia, as palestras serviram para aumentar o conhecimento de 91,8 para 100,0%. Na Tabela 3, percebe-se que a relação entre roer unhas e a transmissão de verminose, também já estava solidificada entre os estudantes, todavia, após as palestras esse percentual foi diminuído. HOLOS, Ano 29, Vol. 2 70 Tabela 3 - Conhecimento sobre o fato de roer e a transmissão de verminoses em alunos da rede pública de ensino nos municípios de Apodi, Felipe Guerra e Severiano Melo no RN, antes e após as palestras (n=677) Momentos Roer unhas influencia quanto a adquirir verminose? Sim Não Não responderam Antes das palestras 88,9% 10,1% 1,0% Após as palestras 72,7% 24,2% 3,0% HOLOS, Ano 29, Vol. 2 70 Tabela 3 - Conhecimento sobre o fato de roer e a transmissão de verminoses em alunos da rede pública de ensino nos municípios de Apodi, Felipe Guerra e Severiano Melo no RN, antes e após as palestras (n=677) Momentos Roer unhas influencia quanto a adquirir verminose? Sim Não Não responderam Antes das palestras 88,9% 10,1% 1,0% Após as palestras 72,7% 24,2% 3,0% Tabela 3 - Conhecimento sobre o fato de roer e a transmissão de verminoses em alu pública de ensino nos municípios de Apodi, Felipe Guerra e Severiano Melo no RN, as palestras (n=677) 70 HOLOS, Ano 29, Vol. 2 70 MOREIRA ET AL. (2013) através de questionários encontraram que 100% dos entrevistados responderam que os vermes dos animais são transmitidos para o homem demonstrando assim conhecimento sobre o potencial zoonótico dos helmintos. Na presente pesquisa com os jovens do RN não foi questionado a forma de transmissão desses vermes, todavia o percentual de 86,5% antes das palestras mostra que o conceito já está estabelecido. através de questionários encontraram que 100% dos entrevistados responderam que os vermes dos animais são transmitidos para o homem demonstrando assim conhecimento sobre o potencial zoonótico dos helmintos. Na presente pesquisa com os jovens do RN não foi questionado a forma de transmissão desses vermes, todavia o percentual de 86,5% antes das palestras mostra que o conceito já está estabelecido. Em várias cidades brasileiras, muitos animais circulam livremente pelas ruas e praças públicas, sejam errantes ou domiciliados conduzidos por seus proprietários. Estes, no momento da defecação, podem contaminar o solo com formas evolutivas infectantes de endoparasitos (Guimarães et al., 2005). Segundo Capuano e Rocha (2005), a crescente aquisição de cães como animais de companhia tem aumentado o número de pessoas expostas ao risco de contrair zoonoses e a população infantil corresponde ao grupo mais exposto devido ao hábito de brincar em contato com o solo e aos hábitos de geofagia, de andar descalço, de se deixar abraçar, lamber e morder por seus animais de companhia. Para Lima et al. (2010), provavelmente, a relação tão próxima do homem com seu animal de estimação seja um fator relevante para preocupação, com formas de evitar que esse convívio não se torne um fator de risco. Tome et al. (2005) citando vários autores e dados de sua própria pesquisa afirmaram que há uma carência de informações por parte das educadoras sobre as zoonoses parasitárias, uma vez que pequenos animais podem manter e disseminar diversos nematóides e protozoários, ocasionando doenças nos seres humanos. Como na presente pesquisa, não ocorreram evoluções significativas nas respostas frente à transmissão, fica claro que esse conceito já estava presente nos alunos e que as palestras não alteraram de maneira significativa o percentual de respostas. Na Tabela 5, tal qual na tabela anterior percebe-se que as palestras não influenciaram de maneira contundente a percepção dos alunos sobre a transmissão de doenças pelas fezes de gato. Na Tabela 4 pode ser observado o conhecimento dos alunos frente à indagação da transmissão de vermes dos animais para os humanos, onde as respostas tiveram valores próximos antes e após as palestras, 86,5 e 87,9%. Tabela 4 - Conhecimento sobre a transmissão de vermes entre animais e seres humanos em alunos da rede pública de ensino nos municípios de Apodi, Felipe Guerra e Severiano Melo no RN, antes e após as palestras (n=677) Momentos Os vermes dos animais são transmitidos para o homem? Sim Não Não responderam Antes das palestras 86,5% 12,7% 0,8% Após as palestras 87,9% 9,1% 3,0% 4 - Conhecimento sobre a transmissão de vermes entre animais e seres humanos em a rede pública de ensino nos municípios de Apodi, Felipe Guerra e Severiano Melo no RN, antes e após as palestras (n=677) Lima et al. (2008) realizaram pesquisas sobre verminose em idosos na cidade de Araçatuba-SP e encontraram que 67,2% dos entrevistados sabiam que os vermes dos animais podem ser transmitidos para o homem. Desses, 34,4% não souberam explicar como ocorre esse processo; 10,0% mencionaram o contato direto com cães e gatos e somente 8,9% citaram a urina e fezes como meio de propagação. Também com idosos, Nunes et al. (2009) ao trabalharem 71 71 HOLOS, Ano 29, Vol. 2 Todavia a percepção sobre a principal doença transmitida pelas fezes melhorou, pois o percentual de respostas associando com a toxoplasmose aumentou de 1,9 para 57,8% após a realização das palestras. Tabela 5 - Conhecimento sobre a transmissão de doenças pelas fezes do gato em alunos da rede pública de ensino nos municípios de Apodi, Felipe Guerra e Severiano Melo no RN, antes e após as palestras (n=677) Momentos As fezes do gato podem transmitir doenças? Sim Não Não responderam Antes das palestras 91,1% 8,1% 0,8% Após as palestras 94,2% 5,8% 0,0% Doenças Se sim, quais doenças as fezes dos gatos podem transmitir? Antes das palestras Após as palestras Toxoplasmose 1,9% 57,8% Outras1 33,3% 21,6% Não respondeu ou esqueceu 35,7% 15,5% Germes 20,4% 3,4% Asma/Alergias/Micoses 8,7% 1,7% 1: Raiva, Calazar, Leptospirose, Salmonelose, etc. onhecimento sobre a transmissão de doenças pelas fezes do gato em alunos da de ensino nos municípios de Apodi, Felipe Guerra e Severiano Melo no RN, antes e t ( 677) Tabela 5 - Conhecimento sobre a transmissão de doenças pelas fezes do gato em alunos da rede pública de ensino nos municípios de Apodi, Felipe Guerra e Severiano Melo no RN, antes e após as palestras (n=677) 72 72 HOLOS, Ano 29, Vol. 2 MOREIRA ET AL. (2013) Em pesquisas de percepção sobre a Toxoplasmose, Tome et al. (2005) encontraram que 92,24% de professoras do ensino infantil associaram o felino como transmissor da toxoplasmose. Em pesquisa realizada por Lima et al. (2008) em idosos a respeito da infecção toxoplásmica, 78,4% ignoravam completamente o assunto e 86,6% não souberam indicar possíveis formas de transmissão da doença. Segundo Araújo e Teixeira (2010), a toxoplasmose também é conhecida como doença do gato, acomete todos os vertebrados de sangue quente, onde as formas de transmissão para os seres humanos são a ingestão de cistos em carnes mal cozidas, oocistos em água contaminada, ou na forma congênita. Ainda de acordo com os mesmos autores, os felinos infectam-se com toxoplasmose por ingestão de tecidos de roedores ou de carne crua de outras espécies animais. A chave da epidemiologia da toxoplasmose parece ser o gato de rua que ao defecar no ambiente contamina a areia e solo e tornam-se fontes duradouras de infecção. Dependendo das condições ambientais, o agente da toxoplasmose pode permanecer infectante por até 18 meses. MOREIRA ET AL. (2013) analisou a relação de casos de leptospirose e índices pluviométricos em Fortaleza-CE entre 2001 e 2004, e encontraram que a maior incidência ocorreu no primeiro semestre, semelhante às precipitações de chuva. analisou a relação de casos de leptospirose e índices pluviométricos em Fortaleza-CE entre 2001 e 2004, e encontraram que a maior incidência ocorreu no primeiro semestre, semelhante às precipitações de chuva. Almeida et al. (2009), trabalhando com professores e agentes comunitários encontraram que 100% das duas categorias associaram a leptospirose com a urina de ratos. Quanto à transmissão da leptospirose pela água contaminada, 91,6% dos professores e 85% dos agentes identificaram como positiva essa associação. Aleixo e Sant’Anna Neto (2011) trabalharam com cidadãos de Ribeirão Preto-SP de áreas endêmicas para doenças de veiculação hídrica e com relação à leptospirose, a maioria dos participantes (60%) disse não saber como se prevenir. De maneira contrária, Coiro et al. (2011) realizaram trabalho de levantamento do nível de conhecimento sobre a leptospirose de agentes de saúde em Botucatu-SP e de uma maneira geral os agentes foram capazes de detectar os principais fatores de risco relacionados à doença no homem. A região de Apodi e Felipe Guerra-RN possui uma grande produção de arroz, cultura agrícola que pode suscitar a presença da leptospirose. Barcellos et al. (2003) relataram casos de plantadores de arroz contaminados por leptospirose. Segundo esses autores, supõe-se que a possível rota de transmissão da leptospirose tenha sido o contato prolongado dos pacientes com a água contaminada da plantação de arroz, caracterizando uma exposição ocupacional. De modo a prevenir a leptospirose uma alternativa seria a vacinação, contudo ainda não existem vacinas disponíveis para o homem. Dessa forma, segundo Balassiano et al. (2011), deveriam ser implementadas medidas de proteção individual aos trabalhadores. Nas áreas com risco de leptospirose, é preciso implantar projetos desenvolvidos com uma antecedência mínima de três meses do período de chuvas, investindo-se sempre na integração dos diferentes órgãos governamentais, associações de moradores e ONGs que possam minimizar ou eliminar as condições de risco (Brasil, 2002a). Na tabela 7 é observado o percentual de presença de ratos nas residências dos alunos, onde 31,1% dos domicílios possuem esses roedores, que constituem no principal transmissor da Leptospirose. Nesse sentido, o solo contaminado com fezes dos gatos domésticos é uma via de transmissão de grande importância epidemiológica, mas o contato com o animal não resulta grande perigo porque os oocistos não se aderem aos pelos do gato. O fato dos alunos já possuírem o conceito da relação de transmissão entre fezes de gato e doenças é importante, todavia o fato de não se saber qual a doença transmitida é preocupante. Dessa forma, as palestras foram importantes, pois conseguiram aumentar o aprendizado dos alunos, no entanto é necessário que as escolas desenvolvam trabalhos contínuos para que o conhecimento seja solidificado. Na tabela 6 está exposto o conhecimento dos alunos sobre a transmissão de leptospirose, onde o percentual de respostas afirmativas aumentou de 29,4 para 72,4% após as palestras. Tabela 6 - Conhecimento sobre a transmissão de Leptospirose em alunos da rede pública de ensino nos municípios de Apodi, Felipe Guerra e Severiano Melo no RN, antes e após as palestras (n=677) Tabela 6 - Conhecimento sobre a transmissão de Leptospirose em alunos da rede pública de ensino nos municípios de Apodi, Felipe Guerra e Severiano Melo no RN, antes e após as palestras (n=677) Momentos Você sabe como se pega a Leptospirose? Sim Não Não responderam Antes das palestras 29,4% 70,6% 0,0% Após das palestras 72,4% 26,9% 0,7% A leptospirose constitui-se em uma doença que aumenta a importância em períodos chuvosos ou de enchentes, com isso os trabalhos educativos realizados pelos agentes de saúde e ou escolas possuem maior concentração no período de inverno. Isso pode ser evidenciado no percentual de alunos que responderam afirmativamente frente a transmissão da leptospirose antes das palestras (29,4%), uma vez que a leptospirose não é uma doença trabalhada ao longo do ano e sim nos períodos considerados críticos. Segundo Costa et al. (2001), a leptospirose está associada a populações de baixo nível socioeconômico, com deficiência de saneamento básico, com dificuldade de acesso a educação e saúde, habitando moradias precárias, em regiões periféricas, às margens de córregos ou esgotos a céu aberto e expostos com frequência a enchentes. Morikawa (2010) reforça que a leptospirose só será efetivamente controlada com a melhoria das condições de moradia e de saneamento ambiental, principalmente nos períodos chuvosos, nesse sentido, Moreira (2006) HOLOS, Ano 29, Vol. 2 73 MOREIRA ET AL. (2013) capacidade adaptativa e comportamento oportunista, os roedores tem sido um dos grupos mais competentes na colonização de novas áreas urbanas. Esses mesmos autores analisaram a infestação por roedores em Salvador e identificaram que 67,4% dos domicílios visitados apresentaram um ou mais sinais de infestação por roedores através de presença de tocas (49,3%), trilhas (47,1%) e fezes (45,7%). No trabalho realizado com os alunos não foram questionados possíveis sinais de infestação, dessa forma, o índice de 31,1% encontrado pode ser subavaliado em virtude da investigação não ter sido mais aprofundada. Na tabela 8, pode ser observada as formas de combate aos roedores nas residências do alunos da rede pública de ensino, onde os principais resultados foram que 48,3% usam venenos e 18,4% usam ratoeiras. Tabela 8 - Levantamento sobre as principais formas de combate a ratos nas residências de alunos da rede pública de ensino nos municípios de Apodi, Felipe Guerra e Severiano Melo no RN (n=396) Como os ratos são combatidos? Uso de veneno 43,3% Uso de ratoeiras 18,4% Gatos 12,8% Não respondeu 9,8% Mata os ratos 8,5% Nada faz 3,5% Outros1 3,5% 1: limpeza do ambiente e uso de cães. evantamento sobre as principais formas de combate a ratos nas residências de de pública de ensino nos municípios de Apodi, Felipe Guerra e Severiano Melo no Tabela 8 - Levantamento sobre as principais formas de combate a ratos nas residências de alunos da rede pública de ensino nos municípios de Apodi, Felipe Guerra e Severiano Melo no RN (n=396) Tabela 8 - Levantamento sobre as principais formas de combate a ratos nas residências de alunos da rede pública de ensino nos municípios de Apodi, Felipe Guerra e Severiano Melo no RN (n=396) Segundo Brasil (2002b), surtos epidêmicos de Leptospirose humana, bem como atendimentos efetivados nas unidades de saúde podem servir como justificativa relevante para implantação de um programa de controle de roedores, principalmente se estes agravos se tornarem constantes e em número crescente. Conforme Brasil (2002b) e Machado et al. (2010), a remoção ou limitação das fontes de alimentos disponíveis para os ratos, por exemplo, é uma medida de manejo integrado. Diminuir ou mesmo suprimir-lhes as fontes de água, é outro fator controlador. Eliminar possíveis abrigos das espécies-alvo pode criar dificuldades intransponíveis para alguns roedores. Tabela 7 - Percentual de presença de ratos nas residências de alunos da rede pública de ensino nos municípios de Apodi, Felipe Guerra e Severiano Melo no RN (n=396) A sua casa possui ratos? Sim Não 31,1% 68,9% Tabela 7 - Percentual de presença de ratos nas residências de alunos da rede públi nos municípios de Apodi, Felipe Guerra e Severiano Melo no RN (n=396) A i t ? A presença de ratos desde a antiguidade é motivo de relatos, segundo Ávila-Pires (1989) há mais de 1000 anos os povos orientais associavam as epidemias de peste bubônica à presença de ratos e a primeira referência segura provém da Bíblia. Para Morikawa (2010), no Brasil, acredita-se que a maioria dos casos urbanos de Leptospirose possua o rato domestico como principal reservatório. Nos centros urbanos, a deficiência de saneamento básico constitui um fator essencial para a proliferação de roedores. Portanto, os grupos socioeconômicos menos privilegiados são os que apresentam maior risco de contrair a infecção. Segundo Veras et al. (1987), a média de ratos em bairros de classe rica é de 2-3 ratos por habitante, enquanto em bairros pobres e favelas a concentração pode chegar a 4-6 ratos por habitante. acordo com Santos et al. (2009), por apresentar elevada taxa de reprodução, alt 74 HOLOS, Ano 29, Vol. 2 74 CONCLUSÕES O presente trabalho evidenciou que as palestras alcançaram o objetivo de passar informações úteis para os alunos, com melhoria na aprendizagem e com fixação de conceitos de transmissão e prevenção de algumas zoonoses, todavia ainda há a necessidade de trabalhos educativos sobre zoonoses com os jovens, para que os mesmos atuem como multiplicadores. O diagnóstico realizado pode servir de base para que as escolas participantes executem trabalhos contínuos de conscientização dos alunos sobre Zoonoses. O diagnóstico realizado pode servir de base para que as escolas participantes executem trabalhos contínuos de conscientização dos alunos sobre Zoonoses. MOREIRA ET AL. (2013) aquelas que capturam os roedores vivos (incruentas) e há as que produzem sua morte no ato de captura (cruentas). Dentre estas últimas, a mais difundida em todo o mundo é a popular ratoeira “quebra-costas” cujas origens remontam a épocas medievais (Brasil, 2002b). aquelas que capturam os roedores vivos (incruentas) e há as que produzem sua morte no ato de captura (cruentas). Dentre estas últimas, a mais difundida em todo o mundo é a popular ratoeira “quebra-costas” cujas origens remontam a épocas medievais (Brasil, 2002b). O uso de gatos também foi citado pelos entrevistados, todavia, na prática, apenas os gatos rueiros ainda conservam seus instintos plenos e os exercitam constantemente. Porém, utilizá-los de forma maciça como método de controle da população murina, é medida arriscada em virtude do próprio potencial de transmissão zoonótica que os felinos domésticos possuem, ou seja, eles são capazes de transmitir doenças à espécie humana como a raiva e a toxoplasmose (Brasil, 2002b). Segundo Santos et al. (2009), a seleção e aplicação dos métodos de controle de roedores para prevenção da leptospirose urbana dependem das características ambientais específicas e das espécies alvo presentes. Estudos ecológicos urbanos que identifiquem as espécies predominantes de roedores, assim como os fatores ambientais que determinam a sua presença são necessários para o planejamento de medidas de intervenção, prevenindo leptospirose e diminuindo o impacto ambiental. Conforme Brasil (2002a), a prevenção de leptospirose é realizada pelo controle da população de roedores, redução de riscos de exposição às águas de enchentes, uso de equipamentos de proteção individual por trabalhadores que apresentam riscos de contaminação, uso de hipoclorito de sódio para desinfecção de áreas contaminadas, distribuição de água potável, vigilância sanitária dos alimentos, destino adequado do lixo, desassoreamento, limpeza e canalização de córregos e vacinação de animais. Esse trabalho contou com o apoio irrestrito por parte das escolas, tal qual observado por Baltazar et al. (2004), que em pesquisa com professores das escolas municipais de São Paulo encontrou grande aceitação por parte das coordenações pedagógicas das escolas, comprovando- se, assim, a necessidade e utilidade de se estabelecer uma ligação entre a universidade (fonte de pesquisa e novos conhecimentos) e professores do ensino fundamental que podem trabalhar como multiplicadores junto à comunidade que já tem tantos problemas a solucionar. Em suma, toda e qualquer medida que lhes dificultem a vida pode, por si só, causar um impacto bastante forte nas populações de roedores. Se a esse manejo for adicionado uma forma de combate direto (métodos químicos e/ou físicos) buscando eliminar os roedores já existentes, a resultante será um controle mais prolongado, eventualmente até permanente do problema. No presente trabalho, o uso dos venenos raticidas foi a forma de controle mais utilizada. O controle químico é praticado através de substâncias naturais ou sintéticas, capazes de provocar a morte dos roedores que as ingerirem. São chamadas genericamente de raticidas em nosso país, ainda que o termo correto devesse ser rodenticidas (Brasil, 2002b). O uso desses produtos em cidades do interior é realizado de maneira indiscriminada e com pouca ou nenhuma fiscalização, sendo utilizadas, inclusive, substâncias proibidas, como a estricnina, o monofluoracetato e o aldicarb. Outra forma citada como controle pelos alunos da rede pública de Apodi, Felipe Guerra e Severiano Melo foram as ratoeiras. Desde que o homem tornou-se caçador, as ratoeiras ou armadilhas são empregadas. Há uma infinidade literalmente ilimitada de tipos e conceitos. Há 75 HOLOS, Ano 29, Vol. 2 75 MOREIRA ET AL. (2013) 3. ALMEIDA FILHO, N.; ROUQUAYROL, M. Z. Introdução à epidemiologia. 3. ed. Rio de Janeiro: MEDSI, 2002, 293 p. 3. ALMEIDA FILHO, N.; ROUQUAYROL, M. Z. Introdução à epidemiologia. 3. ed. 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BRASIL. Ministério da Saúde. Vigilância Epidemiológica, 2011. 14. BRASIL. Ministério da Saúde. Vigilância Epidemiológica, 2011. 15. CAPUANO D.M., ROCHA G.M. Environmental contamination by Toxocara sp eggs in Ribeirão Preto, São Paulo State, Brazil.Rev. Inst. Med. Trop.,v.47, n.4, p.223-226, 2005. 16. COIRO C.J. et al. Fatores de risco para leptospirose, leishmaniose, neosporose e toxoplasmose em cães domiciliados e peridomíciliados em Botucatu-SP. Vet. REFERÊNCIAS BIBLIOGRÁFICAS 1. ALEIXO, N. C. R.; SANT’ANNA NETO, J. L. Percepção e riscos, abordagem socioambiental do processo saúde-doença. Mercator, v. 10, n. 22, p. 191-208, 2011. 2. ALMEIDA, J. C. et al. Percepção sobre Zoonoses de Professores e Agentes Comunitários de Saúde do Município de Quatro Barras PR. 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Percepção sobre Zoonoses de Professores e Agentes Comunitários de Saúde do Município de Quatro Barras PR. In: IV CONGRESSO LATINO AMERICANO, X CONGRESSO BRASILEIRO DE HIGIENISTAS DE ALIMENTOS, III ENCONTRO DE CENTROS DE CONTROLE DE ZOONOSES E II ENCONTRO DO SISTEMA BRASILEIRO DE INSPEÇÃO DE PRODUTOS DE ORIGEM ANIMAL., 2009, Florianopólis. Anais... Revista Higiene Alimentar. São Paulo : DPI Estudio e Editora Ltda., 2009. v. 23. p. 602-603. 76 HOLOS, Ano 29, Vol. 2 76 MOREIRA ET AL. (2013) 21. GUIMARÃES, A. M, ALVES, E.G.L., REZENDE, G.F., RODRIGUES, M.C. Ovos de Toxocara sp. e larvas de Ancylostoma sp. em praça pública de Lavras, MG.Revista de Saúde Pública, v. 39 n. 2, p. 293-295, 2005. 22. LANGONI, H. Zoonoses and human beings. Journal of Venomous Animal and Toxins including Tropical Diseases, v.10, n.2, p. 111-111. 2004. 23. LIMA, F. F.; KOIVISTO, M. B.; PERRI, S. H. V.; BRESCIANI; K. D. S.; O conhecimento de idosos sobre parasitoses em Instituições não governamentais do município de Araçatuba, SP.Rev. Ciênc. Ext. v.4, n.1, p.83, 2008. 24. LIMA, A.M.A., ALVES, L.C., FAUSTINO, M.A.G., LIRA, N.M.S. Percepção sobre o conhecimento e profilaxia das zoonoses e posse responsável em pais de alunos do pré-escolar de escolas situadas na comunidade localizada no bairro de Dois Irmãos na cidade do Recife (PE).Ciência & Saúde Coletiva, v. 15, Supl. 1, p.1457-1464, 2010. 25. MACHADO, F. M. E.; COELHO, H. E.; REZENDE, R. S. Plano de ação para o controle da leptospirose no Zoológico municipal de Uberaba-MG.Biosci. J.,v. 26, n. 6, p. 981-989, 2010. 26. MINISTÉRIO DA SAÚDE.Situação epidemiológica das zoonoses de interesse para a saúde pública.Boletim eletrônico Epidemiológico, Ano 10, n. 2, 2010. Disponível em: www.saude.gov.br/svs Acesso em: 27 nov 2011. 27. MOREIRA, J.O. Leptospirose humana no município de Fortaleza: abordagens sócio- ambiental. 2006. 100 p.Dissertação - Universidade Federal do Ceará, 2006. 28. MORIKAWA, V.M. Leptospirose. In: Programa de Zoonoses região Sul. Manual de Zoonoses. Curitiba-PR: 2ª ed., v. 1, 2010, p.91-99. 29. NUNES, E.R.C., ALMEIDA, D.B., GONÇALVES, M.A., SILVA, M.R. et al. Percepção dos idosos sobre o conhecimento e profilaxia de zoonoses parasitárias. In: IX JORNADA DE ENSINO, PESQUISA E EXTENSÃO (IX JEPEX), UFRPE, 3p. 2009 30. OLIVEIRA, E.A.; SOUZA, P.S.; SILVA, M.C.P.; et al. Educação de crianças para um convívio pacífico com morcegos e prevenção da Raiva no município de Guaraqueçaba – Paraná–Brasil. Universidade Federal do Paraná. Disponível em: http://www.zoonoses.agrarias.ufpr.br/outraspubl/2008_CCZ_AUGM.pdf. Acesso em: 21/09/09. 31. OLIVEIRA, W.S., CONCEIÇÃO, A.M., BASTOS, N.M.A., SOARES, C.R.P. et al.Avaliação do conhecimento prévio dos alunos ingressantes no curso de licenciatura plena em ciência biológicas da UFRPE sobre zoonoses. In: X JORNADA DE ENSINO, PESQUISA E EXTENSÃO (X JEPEX) UFRPE, 3p., 2010. 32. SANTOS, N. et al.Infestação por roedores no ambiente urbano: o papel das deficiências ambientais na transmissão da leptospirose.In: III CONGRESSO LATINO AMERICANO DE ECOLOGIA, 2009, São Lourenço – MG. Anais... São Lourenço – MG, 2009, 4 p. e Zootec., v.18, n.3, p. 393-407, 2011. 17. CORDEIRO, T.G.P., MACEDO, H.W. Amebíase.Revista de patologia tropical, v. 36, n.2, p. 119-128, 2007. 18. COSTA, E. et al.Formas graves de leptospirose: aspectos clínicos, demográficos e ambientais.Revista da Sociedade Brasileira de Medicina Tropical, v.34, n.3, p. 261-267, 2001. 19. COSTA, S.S., SILVA, B.F.P., MORAIS, A.F.C., WANDERLEY, F.S.Ocorrência de parasitas intestinais em material subungueal e fecal em crianças de uma creche no município de Maceió – Alagoas. Pediatria, v.31, n.3, p.198-203, 2009. 20. FENSTERSEIFER, L.M., WIEST, J.M. Autocuidado em Zoonoses (Nota prévia).Arq. Fac. Vet. UFRGS, v. 14, p. 17-21, 1986. HOLOS, Ano 29, Vol. 2 77 77 33. SILVA NETO, E.J., MANGUEIRA, S.O., FREITAS, S.V. et al.Educação popular como meio de prevenção de zoonoses. In: Congresso Brasileiro de Extensão Universitária, João Pessoa:EDUFPB, 5 p., 2002. 34. TOME, R. O.; SERRANO, A. C. M.; NUNES, C. M.; PERRI, S. H. V.; BRESCIANI, K. D. S. Inquérito epidemiológico sobre conceitos de zoonoses parasitárias para professores de escolas municipais do ensino infantil de Araçatuba-SP.Rev. Ciênc. Ext. v.2, n.1, p.46, 2005. 35. VERAS, F.M.F., ROUQUAYROL, M.Z., GOMES, I.L.P. Estudo epidemiológico dos casos de leptospirose registrados em Fortaleza na epidemia de 1985. Rev. Med. Univ. 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https://openalex.org/W2559171720	https://www.nature.com/articles/ncomms13621.pdf	English	null	Quantifying electronic band interactions in van der Waals materials using angle-resolved reflected-electron spectroscopy	Nature communications	2,016	cc-by	6,788	ARTICLE Received 19 Jul 2016 \| Accepted 17 Oct 2016 \| Published 29 Nov 2016 Received 19 Jul 2016 \| Accepted 17 Oct 2016 \| Published 29 Nov 2016 1 Huygens-Kamerlingh Onnes Laboratorium, Leiden Institute of Physics, Leiden University, Niels Bohrweg 2, P.O. Box 9504, NL-2300 RA Leiden, The Netherlands. 2 Department of Physics, Columbia University, New York, New York 10027, USA. 3 Departamento de Fı´sica de Materiales, Universidad del Pais Vasco UPV/EHU, 20080 San Sebastia´n/Donostia, Spain. 4 IKERBASQUE, Basque Foundation for Science, E-48013 Bilbao, Spain. 5 Donostia International Physics Center (DIPC), E-20018 San Sebastia´n, Spain. 6 IBM T.J. Watson Research Center, 1101 Kitchawan Road, P.O. Box 218, Yorktown Heights, New York, New York 10598, USA. Correspondence and requests for materials should be addressed to J.J. (email: jobst@physics.leidenuniv.nl). ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13621 novel technique offers high lateral resolution, allowing us to measure band structures on small, exfoliated ﬂakes. Although the optimal lateral resolution of ARRES is B10nm (ref. 16), here we integrate over larger areas to improve the signal-to-noise ratio. ARRES uses the fact that the reﬂectivity of low-energy electrons depends strongly on both their kinetic energy E0 (refs 14,15,19) and their in-plane momentum k\|\| (refs 20,21) (both of which can be precisely tuned in LEEM). In particular, the electron reﬂection probability for a speciﬁc combination of E0 and k\|\| is high when the material studied has a band gap at that energy (red in Fig. 1a,b). The reﬂection probability is low, in contrast, if E0 and k\|\| coincide with an unoccupied free-electron-like band (blue in Fig. 1a,b). The special case k\|\|¼ 0 (normal electron incidence) is regularly used in so-called LEEM-IV experiments22,23. It is noteworthy that we cannot measure between the Fermi level (maximum for ARPES) and the vacuum energy (minimum for ARRES). The resulting gap, the work function, is incorporated to scale in Fig. 1. In total, the ARPES–ARRES combination gives insight into an exceptionally wide energy range of the band structure. In fact, in Fig. 1a,b, all bands around the G-point are probed, except for the lower edge of the conduction band. Moreover, the combined data are well- described by band structure calculations for bulk graphite and hBN (black lines in Fig. 1a,b, respectively). E lectronic band structure is the key to most properties of crystalline materials. Band structure measurements are therefore widely used to study the subtle interplay of electrons with lattice excitations1,2 or collective electron phenomena3. The bands and their dispersions originate from the quantum overlap between the electronic states of the atoms that make up the crystal. Consequently, the coupling between the electron systems of the individual layers of van der Waals (vdW) materials4 is encoded in their band structure5,6. Heterostructures of graphene and hexagonal boron nitride (hBN) are widely used to screen electrons in graphene from the environment, therefore providing high electron mobility7–9. Isolation of the two materials is generally assumed over the full energy range, although small changes in the graphene band structure are observed as a function of the stacking angle of graphene and hBN10–13. Results d Band structure of the bulk materials. Figure 1a,b show local measurements on mechanically exfoliated graphite and bulk hBN ﬂakes of B20 nm thickness, respectively. The occupied bands (negative energies in Fig. 1a,b) are measured with ARPES using a helium ultraviolet light source18, whereas the unoccupied bands (positive energies in Fig. 1a,b) are studied using ARRES16. This E0 (eV) E0 (eV) E0 (eV) E0 (eV) E0 (eV) E0 (eV) 0 5 10 15 20 25 30 35 40 M K Γ 1.0 0.5 0.0 0.5 1.0 1.5 k\|\| (Å–1) k\|\| (Å–1) k\|\| (Å–1) k\|\| (Å–1) k\|\| (Å–1) k\|\| (Å–1) k\|\| (Å–1) k\|\| (Å–1) 1.0 0.5 0.0 0.5 1.0 –20 –15 –10 –5 M M Γ σ π 0 5 10 15 20 25 30 35 40 1.0 0.5 0.0 0.5 1.0 M M Γ σ π 1.0 0.5 0.0 0.5 1.0 1.5 M K Γ –20 –15 –10 –5 0 5 10 15 20 25 30 35 40 1.0 0.5 0.0 0.5 1.0 1.5 1.0 0.5 0.0 0.5 1.0 M K Γ 0.01 0.5 0.1 Intensity (a. u.) 0 5 10 15 20 25 30 35 40 1.0 0.5 0.0 0.5 1.0 1.5 1.0 0.5 0.0 0.5 1.0 M K Γ –20 –15 –10 –5 b d c a Figure 1 \| Measured band structures of the mother compounds graphite and bulk hBN. (a) Band structure of graphite. The unoccupied bands (positive energies) are measured by ARRES, the occupied bands (negative energies) by ARPES. The full band structure is well described by band structure calculations (black lines, adapted from ref. 27). (b) Experimental band structure of bulk hBN measured as in a. Black lines are calculated band edges. (c) ARRES spectrum calculated with a full-potential linear augmented plane waves method (Methods section and refs 24–26). (d) Calculated projected density of states of bulk hBN. The features in the measured unoccupied band structure in b can easily be identiﬁed with bands and band gaps, as well as ﬁne pockets in the calculations (for example, arrows in d). ARTICLE E g g g p Here we scrutinize the band structure of graphene–hBN heterostructures over a wide energy range to shed light on the interactions in this most widely used vdW system. We apply a series of experimental techniques based on low-energy electron microscopy (LEEM) to assess structural and electronic properties in situ, with high lateral resolution14,15. First, we study the band structures of graphite and bulk hBN as a reference. Combining LEEM-based angle-resolved photoemission spectro- scopy (ARPES) and angle-resolved reﬂected-electron spectro- scopy (ARRES)16, we deduce information on both the occupied and unoccupied bands over an unprecedented energy range. Then we investigate the band evolution in few-layer hBN and show that our ARRES data match very well with ab initio calculations. Finally, we turn to stacks of few-layer graphene on hBN to study their electronic coupling over an energy range of B25 eV. All samples are produced on a conductive silicon substrate using a polymer-free assembly technique17 to guarantee clean surfaces and graphene–hBN interfaces (see Methods section). g p y Interestingly, ARRES does not only reveal band edge positions. Rather, ARRES probes the full transmission states of a material. Those states can be found by calculating the full electron scattering problem starting from a plane wave in the vacuum half-space above the sample and computing its reﬂection and transmission at the slab of material (see details in the Methods section and refs 24–26). A calculated ARRES spectrum for bulk hBN is plotted in Fig. 1c and shows striking similarity to the measurements in Fig. 1b. Most intuitively, ARRES data can be compared with the density of unoccupied states projected onto the sample plane. To illustrate this, we calculate the projected density of states for bulk hBN from its Kohn–Sham band structure in the local density approximation24. Remarkably, this calculation shown in Fig. 1d does not only describe the large band gaps (observed as red areas in Fig. 1b) but also the subtle pockets in the band structure (for example, the gaps marked with arrows in Fig. 1d). The energy resolution of our LEEM microscope is B150 meV, which determines the minimal features of the band structure that can be probed in our instrument. It is worth noting Quantifying electronic band interactions in van der Waals materials using angle-resolved reﬂected-electron spectroscopy Johannes Jobst1,2, Alexander J.H. van der Torren1, Eugene E. Krasovskii3,4,5, Jesse Balgley2, Cory R. Dean2, Rudolf M. Tromp1,6 & Sense Jan van der Molen1 Johannes Jobst1,2, Alexander J.H. van der Torren1, Eugene E. Krasovskii3,4,5, Jesse Balgley2, Cory R. Dean2, Rudolf M. Tromp1,6 & Sense Jan van der Molen1 Johannes Jobst1,2, Alexander J.H. van der Torren1, Eugene E. Krasovskii3,4,5, Jesse Balgley2, Cory R. Dean2, Rudolf M. Tromp1,6 & Sense Jan van der Molen1 High electron mobility is one of graphene’s key properties, exploited for applications and fundamental research alike. Highest mobility values are found in heterostructures of graphene and hexagonal boron nitride, which consequently are widely used. However, surprisingly little is known about the interaction between the electronic states of these layered systems. Rather pragmatically, it is assumed that these do not couple signiﬁcantly. Here we study the unoccupied band structure of graphite, boron nitride and their heterostructures using angle- resolved reﬂected-electron spectroscopy. We demonstrate that graphene and boron nitride bands do not interact over a wide energy range, despite their very similar dispersions. The method we use can be generally applied to study interactions in van der Waals systems, that is, artiﬁcial stacks of layered materials. With this we can quantitatively understand the ‘chemistry of layers’ by which novel materials are created via electronic coupling between the layers they are composed of. 1 Huygens-Kamerlingh Onnes Laboratorium, Leiden Institute of Physics, Leiden University, Niels Bohrweg 2, P.O. Box 9504, NL-2300 RA Leiden, The Netherlands. 2 Department of Physics, Columbia University, New York, New York 10027, USA. 3 Departamento de Fı´sica de Materiales, Universidad del Pais Vasco UPV/EHU, 20080 San Sebastia´n/Donostia, Spain. 4 IKERBASQUE, Basque Foundation for Science, E-48013 Bilbao, Spain. 5 Donostia International Physics Center (DIPC), E-20018 San Sebastia´n, Spain. 6 IBM T.J. Watson Research Center, 1101 Kitchawan Road, P.O. Box 218, Yorktown Heights, New York, New York 10598, USA. Correspondence and requests for materials should be addressed to J.J. (email: jobst@physics.leidenuniv.nl). 1 NATURE COMMUNICATIONS \| 7:13621 \| DOI: 10.1038/ncomms13621 \| www.nature.com/naturecommunications URE COMMUNICATIONS \| 7:13621 \| DOI: 10.1038/ncomms13621 \| www Results d 2 E0 (eV) E0 (eV) 0 5 10 15 20 25 30 35 40 M K Γ 1.0 0.5 0.0 0.5 1.0 1.5 k\|\| (Å–1) k\|\| (Å–1) 1.0 0.5 0.0 0.5 1.0 –20 –15 –10 –5 M M Γ σ π a E0 (eV) E0 (eV) k\|\| (Å–1) k\|\| (Å–1) 0 5 10 15 20 25 30 35 40 1.0 0.5 0.0 0.5 1.0 M M Γ σ π 1.0 0.5 0.0 0.5 1.0 1.5 M K Γ –20 –15 –10 –5 b E0 (eV) k\|\| (Å–1) k\|\| (Å–1) 0 5 10 15 20 25 30 35 40 1.0 0.5 0.0 0.5 1.0 1.5 1.0 0.5 0.0 0.5 1.0 M K Γ 0.01 0.5 0.1 Intensity (a. u.) c E0 (eV) k\|\| (Å–1) k\|\| (Å–1) 0 5 10 15 20 25 30 35 40 1.0 0.5 0.0 0.5 1.0 1.5 1.0 0.5 0.0 0.5 1.0 M K Γ –20 –15 –10 –5 d d b c a k\|\| (Å–1) k\|\| (Å–1) Figure 1 \| Measured band structures of the mother compounds graphite and bulk hBN. (a) Band structure of graphite. The unoccupied bands (positive energies) are measured by ARRES, the occupied bands (negative energies) by ARPES. The full band structure is well described by band structure calculations (black lines, adapted from ref. 27). (b) Experimental band structure of bulk hBN measured as in a. Black lines are calculated band edges. (c) ARRES spectrum calculated with a full-potential linear augmented plane waves method (Methods section and refs 24–26). (d) Calculated projected density of states of bulk hBN. The features in the measured unoccupied band structure in b can easily be identiﬁed with bands and band gaps, as well as ﬁne pockets in the calculations (for example, arrows in d). NATURE COMMUNICATIONS \| 7:13621 \| DOI: 10.1038/ncomms13621 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13621 curves, thus unambiguously revealing the number of graphene layers27–29. These transmission resonances can be viewed in analogy to a tight-binding model where individual transmission resonances correspond to ‘atomic’ wave functions and are frequently called ‘interlayer states’16,28. It is noteworthy that in contrast to tight-binding theory, the transmission resonances probed here cannot be assigned to localized states and, in particular, are not spatially localized between adjacent layers. In this tight-binding picture, the energetic splitting of the transmission resonances can be interpreted via a hopping integral that quantiﬁes their interaction. Results d Consequently, the energetic separation of the minima in the IV curves is a direct measure for the hopping integral between the individual ‘interlayer states’, that is, their coupling. Interestingly, the same arguments apply to few-layer hBN and hence similar discrete transmission resonances are expected30. To investigate in detail that the almost perfect match between experimental data in Fig. 1b and theoretical predictions in Fig. 1c,d is achieved by ab initio calculations without free parameters. Conversely, ARRES data may serve as a benchmark for more detailed band structure calculations. This is particularly important, as no experimental data have been available in the energy range probed here. Band structure of few-layer hBN. After establishing ARRES as a method to study band properties of graphite and bulk hBN—the mother compounds of vdW heterostructures—let us now discuss these materials in the few-layer limit. It is well known that for few-layer graphene, the continuous conduction band of graphite splits up into quantized transmission resonances. For a stack of n þ 1 graphene layers, one can ﬁnd n such transmission reso- nances, which lead to n characteristic minima in LEEM-IV 0 5 10 15 20 25 0.0 0.2 0.4 0.6 0.8 1.0 Intensity (a.u.) E0 (eV) 2 layers 3 layers 3 layers 4 layers 5 layers Silicon hBN a b c 1 μm 0.06 Intensity (a. u.) 10–3 0.3 0 5 10 15 20 25 E0 (eV) E0 (eV) E0 (eV) E0 (eV) K M K M K M f 0.8 0.4 0 0.4 0.8 2 3 4 5 2 layers 3 layers 4 layers 0.8 0.4 0 0.4 0.8 2 3 4 5 2 layers 3 layers 4 layers e d 0.0 1.0 1.0 0.0 1.0 1.0 0.0 1.0 1.0 k\|\| (Å–1) k\|\| (Å–1) k\|\| (Å–1) k\|\| (Å–1) k\|\| (Å–1) 0 5 10 15 20 25 0.05 0.7 0.3 igure 2 \| Band quantization in few-layer hBN. (a) LEEM micrograph of ﬂakes with different number of hBN layers. E0 ¼ 3.7 eV is used for optimal layer ontrast. (b) IV curves taken by averaging the intensity of the areas indicated in a. Clear minima are observed for 0oE0o6 eV. A number of n minima marked with arrows) corresponds to n quantized interlayer bands and hence, to n þ 1 hBN layers. The IV curves are vertically shifted by 0.1 a.u. for clarity. ARTICLE A PEEM image (right) of the assembled graphene-hBN stack shows clear contrast between the different materials. (b) LEEM-IV curves resolve the thickness-dependent oscillations between 1 eVoE0o5 eV characteristic for few-layer graphene clearly. (c) LEEM images (top and bottom) acquired at E0 ¼ 4.3 eV together with an overlay of the two optical images from a. Comparison of the three images allows it to identify areas with different numbers of graphene layers. The areas where IV curves are taken are marked. The dark lines in the LEEM images are wrinkles in the graphene sheet. 1 eVoE0o5 eV characteristic for few-layer graphene clearly. (c) LEEM images (top and bottom) acquired at E0 ¼ 4.3 eV together with an overlay of the two optical images from a. Comparison of the three images allows it to identify areas with different numbers of graphene layers. The areas where IV curves are taken are marked. The dark lines in the LEEM images are wrinkles in the graphene sheet. of Fig. 3d, respectively). In Fig. 4a, we ﬁt a simple Lambert–Beer law (red lines) to the measured IV curves (black circles) for all samples where the total intensity IvdW reﬂected from the full stack is given by Studying the coupling within heterojunctions. From Fig. 2, we conclude that transmission resonances exist in the same energy range for both few-layer hBN and graphene16,27,28. Consequently, one may expect the corresponding electronic states to interact strongly once the two materials are joined together, especially as the stacking distance is comparable to the interlayer spacing in bulk hBN and graphite. Let us suppose this were true. Then, on stacking few-layer graphene on bulk hBN, the graphene interlayer states should mix with the broad hBN bulk band to form one continuous band. In that case, LEEM-IV curves should exhibit a broad minimum without oscillations for 0 eVoE0o5 eV. However, this scenario is in stark contrast to the experimental results. In Fig. 3b, we show IV curves for several thicknesses of graphene, stacked onto bulk hBN (see Fig. 3a,c). Remarkably, for low energies the IV curves clearly show the oscillations related to the quantization of the graphite band into n transmission resonances for n þ 1 layers, just similar to that for few-layer graphene on Si. Hence, the intimate contact to the layered hBN crystal below does not lead to a common continuous band. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13621 how the band structure of hBN evolves towards the few-layer layer limit, hBN is mechanically exfoliated and few-layer ﬂakes are selected and deposited onto a silicon substrate. Figure 2a shows a bright-ﬁeld LEEM image of a few-layer hBN crystal. In particular, we chose an area where a monolayer hBN ﬂake lies over a natural step edge from trilayer to bilayer hBN as sketched in Fig. 2b. This layer assignment can be made when looking at the LEEM-IV curves in Fig. 2b that are taken at the marked areas in Fig. 2a. Indeed, we observe n minima (marked with arrows in Fig. 2b) for n þ 1 hBN layers, as in few-layer graphene. Clearly, the top monolayer ﬂake adds another quantized transmission state (visible as an additional minimum in the IV curves) to both the three and four hBN layer areas. This indicates that the monolayer ﬂake is electronically coupled to the underlying hBN in the same way these hBN layers are coupled to each other. It is worth noting that the minima expected for E0o2 eV are convoluted with the steep mirror-mode transition and are therefore hard to identify or are completely invisible. b a hBN 10 μm G 10 μm 10 μm 0 5 10 15 20 25 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Bulk BN 4 layers 2 layers 3 layers Intensity (a.u.) E0 (eV) 1 layer b c 1 μm 4 2 3 1 μm hBN 2 3 1 10 μm a hBN 10 μm G 10 μm 10 μm Figure 3 \| Interaction of electronic bands of graphene and hBN. (a) Few-layer graphene is transferred onto a hBN ﬂake (both selected using optical microscopy). A PEEM image (right) of the assembled graphene-hBN stack shows clear contrast between the different materials. (b) LEEM-IV curves resolve the thickness-dependent oscillations between 1 eVoE0o5 eV characteristic for few-layer graphene clearly. (c) LEEM images (top and bottom) acquired at E0 ¼ 4.3 eV together with an overlay of the two optical images from a. Comparison of the three images allows it to identify areas with different numbers of graphene layers. The areas where IV curves are taken are marked. The dark lines in the LEEM images are wrinkles in the graphene sheet. Results d The inset shows a sketch of the ﬂake assembly along the line proﬁle in a. A monolayer hBN ﬂake lies over a step edge from bilayer to trilayer hBN. (c) ARRES measurements performed in the areas of two, three and four hBN layers marked black, red and blue in a, respectively. (d) ARRES calculations for wo, three and four hBN layers. (e) Measured dispersion of the quantized interlayer bands extracted from c. (f) Ab initio calculations describe the dispersion well over the full range of measurement. b 0 5 10 15 20 25 0.0 0.2 0.4 0.6 0.8 1.0 Intensity (a.u.) E0 (eV) 2 layers 3 layers 3 layers 4 layers 5 layers Silicon hBN b a 1 μm a c 0.06 Intensity (a. u.) 10–3 0.3 0 5 10 15 20 25 E0 (eV) E0 (eV) K M K M K M d 0.0 1.0 1.0 0.0 1.0 1.0 0.0 1.0 1.0 k\|\| (Å–1) k\|\| (Å–1) k\|\| (Å–1) 0 5 10 15 20 25 0.05 0.7 0.3 c E0 (eV) 0.8 0.4 0 0.4 0.8 2 3 4 5 2 layers 3 layers 4 layers e k\|\| (Å–1) f E0 (eV) f 0.8 0.4 0 0.4 0.8 2 3 4 5 2 layers 3 layers 4 layers k\|\| (Å–1) e Figure 2 \| Band quantization in few-layer hBN. (a) LEEM micrograph of ﬂakes with different number of hBN layers. E0 ¼ 3.7 eV is used for optimal layer contrast. (b) IV curves taken by averaging the intensity of the areas indicated in a. Clear minima are observed for 0oE0o6 eV. A number of n minima (marked with arrows) corresponds to n quantized interlayer bands and hence, to n þ 1 hBN layers. The IV curves are vertically shifted by 0.1 a.u. for clarity. The inset shows a sketch of the ﬂake assembly along the line proﬁle in a. A monolayer hBN ﬂake lies over a step edge from bilayer to trilayer hBN. (c) ARRES measurements performed in the areas of two, three and four hBN layers marked black, red and blue in a, respectively. (d) ARRES calculations for two, three and four hBN layers. (e) Measured dispersion of the quantized interlayer bands extracted from c. (f) Ab initio calculations describe the dispersion well over the full range of measurement. NATURE COMMUNICATIONS \| 7:13621 \| DOI: 10.1038/ncomms13621 \| www.nature.com/naturecommunications 3 ARTICLE a c 10 μm b 0 5 10 15 20 25 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Bulk BN 4 layers 2 layers 3 layers Intensity (a.u.) E0 (eV) 1 layer b c c 1 μm 4 2 3 1 μm hBN 2 3 1 10 μm Figure 2c shows ARRES measurements performed on bilayer, trilayer and four-layer areas (the size of the used areas is marked by black, red and blue circles in Fig. 2a, respectively). For high energies, these ARRES maps clearly show features identical to bulk hBN (Fig. 1b), conﬁrming that the ﬂakes are few-layer hBN indeed. In addition, we perform ab initio ARRES calculations, that is, solving the full electron scattering problem in the vacuum and crystal half spaces (see Methods). The calculations, shown in Fig. 2d, reproduce our experimental data in almost all details for higher energies and for 0 eVoE0o5 eV as well. The latter is the most exciting part, being the energy range of the quantized quasi- two-dimensional bands of bilayer, trilayer and four-layer hBN. Their respective dispersion relations are deduced from both Fig. 2c (experiment) and Fig. 2d (theory), and are plotted in Fig. 2e,f, respectively. The correspondence between theory (Fig. 2d,f) and experiment (Fig. 2c,e) is excellent without free parameters, demonstrating the strength of ARRES in getting quantitative data on unoccupied bands and their evolution towards the few-layer limit. Figure 3 \| Interaction of electronic bands of graphene and hBN. Figure 3 \| Interaction of electronic bands of graphene and hBN. (a) Few-layer graphene is transferred onto a hBN ﬂake (both selected using optical microscopy). A PEEM image (right) of the assembled graphene-hBN stack shows clear contrast between the different materials. (b) LEEM-IV curves resolve the thickness-dependent oscillations between 1 eVoE0o5 eV characteristic for few-layer graphene clearly. (c) LEEM images (top and bottom) acquired at E0 ¼ 4.3 eV together with an overlay of the two optical images from a. Comparison of the three images allows it to identify areas with different numbers of graphene layers. The areas where IV curves are taken are marked. The dark lines in the LEEM images are wrinkles in the graphene sheet. Figure 3 \| Interaction of electronic bands of graphene and hBN. (a) Few-layer graphene is transferred onto a hBN ﬂake (both selected using optical microscopy). NATURE COMMUNICATIONS \| 7:13621 \| DOI: 10.1038/ncomms13621 \| www.nature.com/naturecommunications Discussion structure measurements31). This is a crucial ﬁnding, as the efﬁcient decoupling of the layers is the underlying reason for the high charge-carrier mobility observed in graphene–hBN heterojunctions. It is worth noting that here we chose thin graphene on top of a thick hBN substrate for its relevance as a widely used system for electronic transport studies. The experimental outcome of ARRES measurements in this two- component heterostack is very clear and can be interpreted straightforwardly. In the more complicated case of few-layer graphene on few-layer hBN, for example, a beating pattern between hBN and graphene transmission resonances emerges32, which bears the risk of obscuring the underlying physics. In these cases, theoretical calculations as shown in Fig. 4b are necessary to understand the IV curves of the combined system. As an example, we show in Fig. 4b how IV curves calculated using a Kronig–Penney model for three layers of graphene on hBN substrates evolve with increasing substrate thickness from 5 to 22 hBN layers. The convergence from a complex beating pattern towards the IV curve of graphene on bulk hBN (as experimentally observed in Fig. 3b) with increasing hBN thickness is apparent in the smoothed calculation (red lines in Fig. 4b). The methodology presented here is thus a versatile tool for studying properties of vdW materials, in particular their interlayer coupling. Combining ARRES with ARPES using a synchrotron source and improving the energy resolution by using an energy-ﬁltered electron source would even allow one to study charge transfer between the layers and determine band offsets in the future. In this letter, we present a test bed for interactions in vdW materials by measuring quantized transmission resonances in their unoccupied band structure. We show that the conduction bands of few-layer hBN and graphene are quantized in a comparable manner. Despite their similar symmetry and energy range, however, the interlayer states of graphene and hBN do not interact electronically. This is an important insight into the graphene–hBN system, which is the most widely used vdW heterostructure. The methods presented here, speciﬁcally ARRES, are directly applicable to more complex vdW materials, for example, stacks including transition-metal dichalcogenides. Consequently, we can study (lack of) electronic overlap between the layers quantitatively. Similar to chemistry, where molecular orbitals are formed from atomic orbitals via their interaction to create compounds with novel properties, this brings us one step closer to a quantitative chemistry of layers. ARTICLE Given the success of this simple Lambert–Beer model together with the existence of the quantized graphene bands on hBN, we can rule out interaction of the interlayer states of few-layer graphene and hBN over an energy range of 30 eV, despite the very similar nature and the intimate contact of the two materials (we note that also at much higher energies, no interaction was observed in near edge X-ray absorption ﬁne NATURE COMMUNICATIONS \| 7:13621 \| DOI: 10.1038/ncomms13621 \| www.nature.com/naturecommunications 4 4 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13621 0.0 0.2 0.4 0.6 0.8 1.0 I (a.u.) I (a.u.) I (a.u.) 0 5 10 15 20 25 E0 (eV) E0 (eV) E0 (eV) E0 (eV) E0 (eV) E0 (eV) 0.0 0.2 0.4 0.6 0.8 1.0 0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 0.0 0.2 0.4 0.6 0.8 1.0 0 5 10 0 5 10 a b 3G-hBN Eq. (1) Ab initio 4G-hBN Eq. (1) Ab initio G-hBN Eq. (1) Ab initio 2G-hBN Eq. (1) Ab initio A B N Bulk hBN Graphite Figure 4 \| Quantitative analysis of the interaction of electronic bands of graphene and hBN. (a) The ﬁrst panel shows IV curves of bulk graphite and hBN. The next panels compare the data in b with two models. Speciﬁcally, we follow the hBN-speciﬁc minimum at E0E8 eV that vanishes continuously as the number of graphene layers on top increases. This behaviour is described well by a simple Lambert–Beer law using equation (1) (red) and by ab initio calculations (light blue). The last panel shows the ab initio result for monolayer graphene on hBN with the carbon atom in the centre of the hBN hexagons (A), above boron atoms (B) and above nitrogen atoms (N). (b) Calculated IV curves for three graphene layers on 5, 9 and 22 hBN layers, respectively. Although for ﬁve hBN layers a beating pattern obscures the signal, the two minima, which are measured for three graphene layers (cf. Fig. 3b) become apparent for a thicker hBN substrate. Black lines are the calculations, red is convoluted with a Gaussian (0.5 eV full width at half maximum) to approximate the experiment. ARTICLE Moreover, if we zoom out to the full energy scale (0–30 eV), we ﬁnd that the IV curves continuously evolve from hBN-like to graphite-like as the number of graphene layers n increases. Speciﬁcally, all hBN-related features (most prominently the deep minimum around E0E8 eV in Fig. 3b) vanish for n ¼ 4. Remarkably, this layer dependence is well-described by a linear combination of the experimental IV curves of graphite, IG(E0), and bulk hBN, IhBN(E0) (green and blue curves in the ﬁrst panel IvdW E0; n ð Þ¼IG E0 ð Þ þ IhBN E0 ð Þ s e n=d: ð1Þ ð1Þ The ﬁt works remarkably well for all n and the full energy range (it is noteworthy that the layer-dependent low-energy minima cannot be reproduced with this approach). We ﬁnd a characteristic penetration depth of d ¼ 0.9 graphene layers and a ratio of interaction cross-sections of low-energy electrons with graphene and hBN of s ¼ 15. Moreover, this simple ﬁt describes the data at least as well as more sophisticated electron reﬂection calculations that take band structures and coherent effects into account (light blue lines). The last panel in Fig. 4a shows ab initio calculations of IV curves for different stacking geometries of graphene on hBN. The result is independent of the stacking geometry, which suggests that the unoccupied bands are not signiﬁcantly affected by the details of the Moire´ superlattice formed due to the lattice mismatch between graphene and hBN, a subtle effect that can be resolved at low temperatures close to the Fermi level10–13. Discussion Detailed insight into the interlayer coupling marks an important step towards creating vdW heterostructures with properties not available in conven- tional materials. ARTICLE 0.0 0.2 0.4 0.6 0.8 1.0 I (a.u.) I (a.u.) 0 5 10 15 20 25 E0 (eV) E0 (eV) E0 (eV) 0.0 0.2 0.4 0.6 0.8 1.0 0 5 10 15 20 25 0 5 10 15 20 25 a 3G-hBN Eq. (1) Ab initio 4G-hBN Eq. (1) Ab initio G-hBN Eq. (1) Ab initio 2G-hBN Eq. (1) Ab initio A B N Bulk hBN Graphite a I (a.u.) E0 (eV) E0 (eV) E0 (eV) 0 5 10 0.0 0.2 0.4 0.6 0.8 1.0 0 5 10 0 5 10 b b I (a.u.) E0 (eV) E0 (eV) E0 (eV) 0 5 10 0.0 0.2 0.4 0.6 0.8 1.0 0 5 10 0 5 10 b Figure 4 \| Quantitative analysis of the interaction of electronic bands of graphene and hBN. (a) The ﬁrst panel shows IV curves of bulk graphite and hBN. The next panels compare the data in b with two models. Speciﬁcally, we follow the hBN-speciﬁc minimum at E0E8 eV that vanishes continuously as the number of graphene layers on top increases. This behaviour is described well by a simple Lambert–Beer law using equation (1) (red) and by ab initio calculations (light blue). The last panel shows the ab initio result for monolayer graphene on hBN with the carbon atom in the centre of the hBN hexagons (A), above boron atoms (B) and above nitrogen atoms (N). (b) Calculated IV curves for three graphene layers on 5, 9 and 22 hBN layers, respectively. Although for ﬁve hBN layers a beating pattern obscures the signal, the two minima, which are measured for three graphene layers (cf. Fig. 3b) become apparent for a thicker hBN substrate. Black lines are the calculations, red is convoluted with a Gaussian (0.5 eV full width at half maximum) to approximate the experiment. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13621 chemical cleaning in chloroform and isopropanol, subsequently. Few-layer hBN is transferred onto silicon substrates using the same method. The measurements presented here do not depend on the substrate used, as long as it is conductive enough to prevent charging. The samples are heated in the LEEM to 500 C, to desorb any residues from the surface. 21. Krasovskii, E. E., Schattke, W., Strocov, V. N. & Claessen, R. Unoccupied band structure of NbSe2 by very low-energy electron diffraction: experiment and theory. Phys. Rev. B 66, 235403 (2002). y y 22. Hannon, J. B., Sun, J., Pohl, K. & Kellogg, G. L. Origins of nanoscale heterogeneity in ultrathin ﬁlms. Phys. Rev. Lett. 96, 246103 (2006). 23. Flege, J. I. & Krasovskii, E. E. Intensity-voltage low-energy electron microscopy for functional materials characterization. Phys. Stat. Solid. Rapid Res. Lett. 8, 463–477 (2014). g y g gy py for functional materials characterization. Phys. Stat. Solid. Rapid Res. Lett. 8, 463–477 (2014). Low-energy electron microscopy. All measurements presented are conducted in the ESCHER setup33, which has a commercially available, aberration-corrected FE LEEM P90 of Specs GmbH as its centrepiece. Microscopy is performed at 5 10 10 mbar and 400 C, to prevent the formation of carbon-based contaminants under the electron beam. Low-energy electron microscopy. All measurements presented are conducted in the ESCHER setup33, which has a commercially available, aberration-corrected FE LEEM P90 of Specs GmbH as its centrepiece. Microscopy is performed at 5 10 10 mbar and 400 C, to prevent the formation of carbon-based contaminants under the electron beam. 24. Krasovskii, E. E., Starrost, F. & Schattke, W. 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K. & Grigorieva, I. V. Van der Waals heterostructures. Nature 499, 419–425 (2013). Acknowledgements 5. Coy Diaz, H. et al. Direct observation of interlayer hybridization and Dirac relativistic carriers in graphene/MoS2 van der Waals heterostructures. Nano Lett. 15, 1135–1140 (2015). We are grateful to Marcel Hesselberth, Daan Boltje and Ruud van Egmond for technical assistance. We thank Charles Kane for fruitful discussions and Kenji Watanabe for supplying the hBN base crystal. This work was supported by the Spanish Ministry of Economy and Competitiveness MINECO (project number FIS2013-48286-C2-1- P) and the Netherlands Organization for Scientiﬁc Research (NWO) via an NWO-Groot grant (‘ESCHER’), a VIDI grant (680-47-502, S.J.v.d.M.), a VENI grant (680-47-447, J.J.) and by the FOM foundation via the ‘Physics in 1D’ programme. C.R.D. acknowledges support from NSF grant DMR-1463465. 6. Le, N. B., Huan, T. D. & Woods, L. M. Interlayer interactions in van der waals heterostructures: electron and phonon properties. ACS Appl. Mater. Interfaces 8, 6286–6292 (2016). 7. Bolotin, K. I., Sikes, K. J., Hone, J., Stormer, H. L. & Kim, P. Tempe- rature-dependent transport in suspended graphene. Phys. Rev. Lett. 101, 96802 (2008). and by the FOM foundation via the ‘Physics in 1D’ programme. C.R.D. acknowledges support from NSF grant DMR-1463465. 8. Du, X., Skachko, I., Barker, A. & Andrei, E. Y. Approaching ballistic transport in suspended graphene. Nat. Nanotechnol. 3, 491–495 (2008). Author contributions 9. Dean, C. R. et al. Boron nitride substrates for high-quality graphene electronics. Nat. Nanotechnol. 5, 722–726 (2010). J.J. designed the experiment, fabricated the samples and performed experiment and data analysis. A.J.H.v.d.T. developed data acquisition and evaluation software. E.E.K. performed band structure and ARRES calculations. J.B. and C.R.D. assisted with sample fabrication. All authors contributed to data interpretation and writing of the manuscript. 10. Dean, C. R. et al. Hofstadter’s butterﬂy and the fractal quantum Hall effect in moire´ superlattices. Nature 497, 598–602 (2013). 11. Hunt, B. et al. Massive Dirac Fermions and Hofstadter Butterﬂy in a van der Waals Heterostructure. Science 340, 1427–1430 (2013). 12. Woods, C. R. et al. Commensurate–incommensurate transition in graphene on hexagonal boron nitride. Nat. Phys. 10, 451–456 (2014). ARTICLE Scattering resonances in two- dimensional crystals with application to graphene. Phys. Rev. B 87, 041405 (R) (2013). 27. Hibino, H. et al. Microscopic thickness determination of thin graphite ﬁlms formed on SiC from quantized oscillation in reﬂectivity of low-energy electrons. Phys. Rev. B 77, 75413 (2008). 28. Srivastava, N. et al. Low-energy electron reﬂectivity of graphene on copper and other substrates. Phys. Rev. B 87, 245414 (2013). Data availability. 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The whole assembly is placed onto a conductive silicon sample via thermal release of the poly(bisphenol A-carbonate) and wet- NATURE COMMUNICATIONS \| 7:13621 \| DOI: 10.1038/ncomms13621 \| www.nature.com/naturecommunications 5 Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ g p g 14. Tromp, R. M. et al. A new aberration-corrected, energy-ﬁltered LEEM/PEEM i t t I P i i l d d i Ult i 110 852 861 (2010) 14. Tromp, R. M. et al. A new aberration-corrected, energy-ﬁltered LEEM/PEEM instrument. I. Principles and design. Ultramicroscopy 110, 852–861 (2010). 15. Tromp, R. M., Hannon, J. B., Wan, W., Berghaus, A. & Schaff, O. A new aberration-corrected, energy-ﬁltered LEEM/PEEM instrument II. Operation and results. Ultramicroscopy 127, 25–39 (2013). How to cite this article: Jobst, J. et al. Quantifying electronic band interactions in van der Waals materials using angle-resolved reﬂected-electron spectroscopy. Nat. Commun. 7, 13621 doi: 10.1038/ncomms13621 (2016). 16. Jobst, J., Kautz, J., Geelen, D., Tromp, R. M. R. M. & Van Der Molen, S. J. S. J. Nanoscale measurements of unoccupied band dispersion in few-layer graphene. Nat. Commun. 6, 8926 (2015). Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 17. Wang, L. et al. One-dimensional electrical contact to a two-dimensional material. Science 342, 614–617 (2013). 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. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Additional information Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. 13. Jung, J., DaSilva, A. M., MacDonald, A. H. & Adam, S. Origin of band gaps in graphene on hexagonal boron nitride. Nat. Commun. 6, 6308 (2015). 18. Tromp, R. M. et al. A simple energy ﬁlter for low energy electron microscopy/ photoelectron emission microscopy instruments. J. Phys. Condens. Matter 21, 314007 (2009). 19. Telieps, W. & Bauer, E. An analytical reﬂection and emission UHV surface electron microscope. Ultramicroscopy 17, 57–65 (1985). 20. 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https://openalex.org/W2338479402	https://zenodo.org/records/1834288/files/article.pdf	English	null	Treatment of Chronic Gonorrhea	The Boston medical and surgical journal/Boston medical and surgical journal	1,901	public-domain	4,509	theim that a urethra becomes tolerant to the gonococ- cus, but that the gonococcus does not lose its virulence when transplanted to a favorable culture medium. vaginitis is most successfully treated by cleansing the vagina with an antiseptic solution and packing it with cotton tampons soaked in medicated solutions, or with dry cotton dusted with an antiseptic or non-irritating powder. In this way the folds are taken out of the mucous membrane, and the applications come into most intimate contact with all parts. As a germi- cide protargol, in solutions of from 1% to 5%, seems to be displacing nitrate of silver as the stand- ard remedy, and has given better results than the many other salts of silver. I have found vaginal suppositories of protargol useful and efficacious, also vaginal suppositories of ichthyol, as' well as the use of the latter drug with glycerin on cotton packings. I th h i transplanted J. Judassohn, of Berne, announces the following conclusions as a result of his study of this question : (1) A mucous membrane affected with chronic gonor- rhea may react by exaggeration of the inflammation when there is a multiplication of the gonococci which it harbors or when new gonococci are introduced ; (2) a mucous membrane affected with chronic gonor- rhea may no longer react in case of multiplication of its own cocci, but may respond by a recrudescence of the inflammation upon the introduction of new cocci ; (3) a mucous membrane affected with chronic gonor- rhea may enjoy a sort of immunity from acute gon- orrhea. He says further that we know nothing of the immunity of the organism on account of the exist- ence of gonorrhea ; even a continued chronic gonor- rhea in one organ does not prevent an acute inflammatory attack in another, for example, acute epididymitis in chronic urethral gonorrhea. W h i g g y packings. In the treatment of chronic urethritis applications to the mucous membrane of the urethra through a small cystoscope of solutions of nitrate of silver, protargol and ichthyol, and also the use of urethral suppositories of the same drugs, have given the best results. Stricture of the urethra is best treated by gradual dilatation with urethral sounds, although rapid dilatation, the patient being under ether, has often given satisfactory results. 1Read before the Boston Society for Medical Improvement, De- cember 31, 1900. The Boston Medical and Surgical Journal as published by The New England Journal of Medicine. Downloaded from nejm.org at UNIVERSITY COLLEGE LONDON on June 28, 2016. For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. Skene's glands are treated by similar applications made with a fine wire appli- cator wound with cotton, and in intractable disease of these glands it is necessary to lay them open into the lumen of the urethra. Th f i i epididymitis g If the following experiments of Wertheim are verified by other observers they will have an impor- tant bearing on the question of the inoculability of gonorrhea. A direct experiment from a pure culture of gonococcus from a gleety discharge of two years' standing gave the following results : (1) Attempted reinfection of the original urethra with this culture was always a failure ; (2) the culture when trans- planted to a coccus-free urethra produced typical acute gonorrhea ; (3) infection from this back again to the original urethra gave a fresh gonorrhea, which, after a typical acute course of five or six weeks, again subsided into a chronic gleet. This explains the fact that an apparently healthy subject of chronic gonor- rhea may infect his hitherto unaffected wife, and be- come again infected from her, that is, the gonococ- cus, by passing through the new culture of the wife, again becomes virulent for the husband. The treatment of cystitis, endometritis, salpingitis and proctitis due to the gonococcus does not differ from the treatment of these affections when caused by other bacteria. Th f h ld y The treatment of gonorrhea should be persisted in until the disease is thoroughly eradicated. The Boston Medical and Surgical Journal as published by The New England Journal of Medicine. Downloaded from nejm.org at UNIVERSITY COLLEGE LONDON on June 28, 2016. For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. BY GARDNER W. ALLEN, M.D., BOSTON. In some cases of gonorrhea a more or less copious, thin, mucopurulent discharge persists for a long time in spite of all efforts to control it. There is an ab- sence of subjective symptoms ; in fact such cases are often mild from the beginning ; the mucous membrane seems flabby and lacking in tone, showing little ten- dency to respond to remedies. These are cases of chronic diffuse urethritis. They are often aggravated by active local measures and over-treatment must be carefully avoided. gIn answer to the question, When should the physi- cian advise marriage after gonorrhea ? Kromayer states that a man may marry when the urethritis is all gone. The gonococcus may be absent, but it is not safe until the discharge has ceased. If the ad- vice is not taken the husband should urinate before coitus, there should be only one coitus in twenty-four hours, and the wife should take a douche after it. Frequent coitus, because of the increased congestion of the sexual organs and the destruction and bruising of the epithelium, increases the danger of infection. ca e u y But, ordinarily speaking, gonorrhea may be said to have reached the chronic stage when the inflammation has become localized at one or more points along the course of the urethra. The discharge, commonly called gleet, is very slight, and the urine is transparent, but contains shreds of mucus and pus. The points at which the subsiding inflammation most commonly lin- gers are the penoscrotal angle and that portion of the pendulous urethra just anterior to it, the bulbomem- branous junction, the prostatic urethra and much less frequently the fossa navicularis. epithelium, danger If we are called upon to pronounce when a woman is entirely free from the danger of transmitting gonor- rhea, it is at once apparent that we have to face a more complicated problem than we have in the case of gonorrhea in the male, because of the many possi- ble lurking places for the gonococcus in the sexual or- gans of the female. As a practical matter we may say that a woman is cured of gonorrhea when there are no signs of chronic inflammation about the uter- ine organs, and when two or more negative cultures have been taken from the urethra and cervical canal. The Boston Medical and Surgical Journal as published by BY GARDNER W. ALLEN, M.D., BOSTON. For the first few days after dilatation, while the folli- cles are still wide open, the patient should use an in- jection of bichloride, silver nitrate or permanganate twice a day. This treatment may be repeated in a week, or the endoscope used, according to circum- stances. In case of stricture this must be gradually dilated at weekly intervals until practically done away with before the endoscope will be of much use. For local applications through the endoscope, nitrate of silver in solutions of a strength of from 3% to 10% gives the best results. In the treatment of these conditions the objects to be aimed at are the removal of fibrous deposits, the restoration of the thickened and rigid mucous mem- brane to approximately its original soft and elastic condition, and the cleaning out of diseased follicles and glands. g a ds The first principle of this treatment is dilatation ; dilatation of the anterior urethra to check the cicatri- cial contraction, cause the absorption of the morbid deposit and open up the follicles so that medicated solutions can penetrate them, and dilatation of the pos- terior urethra in order to open the follicles of the prostate in the same manner and to stimulate circula- tion in the passively congested mucous membrane. p y co gested Ordinary dilatation by means of sounds is often un- satisfactory, chiefly because in many cases it cannot be carried far enough to accomplish results and be- cause it is apt to cause a good deal of irritation and reaction. To cut the meatus sufficiently to admit large sounds is an operation which, although some- times necessary, should be avoided if possible. By means of dilators which can be passed through a mod- erate-sized meatus the urethra can be stretched to a point far beyond what is possible with the largest sounds and yet cause little or no reaction. This dila- tation may be done as gently and gradually as need be at successive visits without any of the discomfort caused by passing a snug-fitting sound through a rigid and unyielding urethra, perhaps painfully distending the meatus at the same time. g In the deep bulbous and prostatic portions the curved dilator is used. When the follicles of the prostate are involved massage is of great assistance. BY GARDNER W. ALLEN, M.D., BOSTON. First, the patient passes his water or part of it, and its appearance is carefully noted, for it furnishes the best guide to the progress of treatment. Then, with some fluid in the bladder, either part of the urine held back or a few ounces of sterilized water injected after micturition, the prostate is thoroughly massaged by the finger in the rectum. The patient now urinates again, entirely emptying his bladder. This urine or fluid is also carefully inspected at each visit, especially with regard to the presence in it of the short, clumpy shreds which come from the follicles of the prostate. Then the posterior dilator is passed and screwed up slowly as far as the patient can bear without discom- fort, left in a few minutes, removed, and an injection of permanganate, silver nitrate or protargol thrown in. This may be repeated once a week. As a rule no treatment is required in the intervals. Wh For the purpose of stretching the urethra the four- branched dilators of Kollmann are the best. A straight instrument is used for the anterior urethra and a curved one for the posterior. In this way only a portion of the urethra is dilated at a time. The urethra is much more tolerant of extreme dilatation at one point than of a less degree applied throughout its length. It is surprising how little pain at the time and how little subsequent reaction are experienced in cases where for special reasons it seems advisable to dilate to what might be considered an extreme degree, that is, to 40 or more. Ordinarily we stop several sizes short of this. After dilatation the local application of various rem- edies is effected by means of injections or irrigations, and later the urethra is inspected through the endo- scope and local applications made under control of the eye to such points as require them. q When the seminal vesicles are involved, stripping or massage of these organs is done in connection with the treatment just described. Sometimes a chronic discharge proceeds from the vesicles alone. In this case treatment is confined to them, and strip- ping is all that is required. BY GARDNER W. ALLEN, M.D., BOSTON. The treatment of gonorrhea in women cannot be considered at length in a paper of this character. It must suffice to say that the treatment varies according to the structures which are attacked. In the more acute stages, when the parts are hypersensitive, douches and extreme cleanliness are to be advised by way of local treatment ; in the chronic stages p , g If we are called upon to pronounce when a woman is entirely free from the danger of transmitting gonor- rhea, it is at once apparent that we have to face a more complicated problem than we have in the case of gonorrhea in the male, because of the many possi- ble lurking places for the gonococcus in the sexual or- gans of the female. As a practical matter we may say that a woman is cured of gonorrhea when there are no signs of chronic inflammation about the uter- ine organs, and when two or more negative cultures have been taken from the urethra and cervical canal. q y The tissue changes following an acute gonorrhea, which determine the localization of the disease at these points, have an important bearing on treatment. The mucous glands or follicles of the urethra are particu- larly numerous about the penoscrotal angle and just anterior to it. They are very liable to be involved in the inflammation together with the tissues about them The treatment of gonorrhea in women cannot be considered at length in a paper of this character. It must suffice to say that the treatment varies according to the structures which are attacked. In the more acute stages, when the parts are hypersensitive, douches and extreme cleanliness are to be advised by way of local treatment ; in the chronic stages 1Read before the Boston Society for Medical Improvement, De- cember 31, 1900. and the entire submucous layer surrounding the urethra, which becomes infiltrated with round cells and shows a tendency to cicatricial contraction. In other words, this is a very common seat of stricture of large calibre complicated with inflammation of the mucous glands. BY GARDNER W. ALLEN, M.D., BOSTON. The submucous layer about the deep bulbous portion of the urethra close to the anterior layer of the triangular ligament is also very subject to deposit of granulation tissue with contraction, and here we are more likely to have strictures of small calibre, either soft and easily dilatable or very dense and tough. In the posterior urethra the tendency to stricture formation does not exist, but the follicles of the prostate may be involved and the inflammation not infrequently extends up the ejaculatory ducts into the seminal vesicles. calibre of the dilators with the rubber cover is about 23 or 24, and it should be possible at any time to use a fair-sized endoscope. If the patient object strongly to meatomy, dilatation may be fairly well done with an Otis urethrotome without the knife. First, an endoscopie examination may be made to aid diagnosis. Eroded or granular patches, inflamed follicles and rigidity of the mucous membrane can be plainly seen. With a bulbous bougie any narrowing, if of sufficient degree, can be detected and located. It is well to learn as much as possible of the topography of the urethra before using the dilator, although this instru- ment also is useful in diagnosis as well as in treat- ment. It is passed well down and opened by slowly and gently turning the screw. Should there be little or no narrowing of the urethral calibre it can be screwed up easily to a high point without pain. When, however, there is stricture a sense of resistance is noticed and considerable force is required in turning the screw and the patient complains of pain. At this point, of course, we stop and either screw down the instrument and remove it or leave it in a few moments. Having removed it, the urethra should be washed out with a solution of corrosive sublimate, potassium per- manganate or anything else that may seem advisable, or we may inject protargol. This drug may be used in these chronic cases in solutions of 10% or even 20% without pain or reaction of consequence, and works extremely well. I generally inject about half a drachm into the bulbous urethra, and while holding the meatus tightly closed, rub it in thoroughly from the outside. BY GARDNER W. ALLEN, M.D., BOSTON. For the first few days after dilatation, while the folli- cles are still wide open, the patient should use an in- jection of bichloride, silver nitrate or permanganate twice a day. This treatment may be repeated in a week, or the endoscope used, according to circum- stances. In case of stricture this must be gradually dilated at weekly intervals until practically done away with before the endoscope will be of much use. For local applications through the endoscope, nitrate of silver in solutions of a strength of from 3% to 10% gives the best results. calibre of the dilators with the rubber cover is about 23 or 24, and it should be possible at any time to use a fair-sized endoscope. If the patient object strongly to meatomy, dilatation may be fairly well done with an Otis urethrotome without the knife. First, an endoscopie examination may be made to aid diagnosis. Eroded or granular patches, inflamed follicles and rigidity of the mucous membrane can be plainly seen. With a bulbous bougie any narrowing, if of sufficient degree, can be detected and located. It is well to learn as much as possible of the topography of the urethra before using the dilator, although this instru- ment also is useful in diagnosis as well as in treat- ment. It is passed well down and opened by slowly and gently turning the screw. Should there be little or no narrowing of the urethral calibre it can be screwed up easily to a high point without pain. When, however, there is stricture a sense of resistance is noticed and considerable force is required in turning the screw and the patient complains of pain. At this point, of course, we stop and either screw down the instrument and remove it or leave it in a few moments. Having removed it, the urethra should be washed out with a solution of corrosive sublimate, potassium per- manganate or anything else that may seem advisable, or we may inject protargol. This drug may be used in these chronic cases in solutions of 10% or even 20% without pain or reaction of consequence, and works extremely well. I generally inject about half a drachm into the bulbous urethra, and while holding the meatus tightly closed, rub it in thoroughly from the outside. The Boston Medical and Surgical Journal as published by The New England Journal of Medicine. Downloaded from nejm.org at UNIVERSITY COLLEGE LONDON on June 28, 2016. For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. The Boston Medical and Surgical Journal as published by The Boston Medical and Surgical Journal as published by Th N E l d J l f M di i D l d d f j t UNIVERSITY COLLEGE LONDON J 28 2016 g p y The New England Journal of Medicine. Downloaded from nejm.org at UNIVERSITY COLLEGE LONDON on June 28, 2016. For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. The Boston Medical and Surgical Journal as published by Medicine. Downloaded from nejm.org at UNIVERSITY COLLEGE LONDON on June 28, 2016. ith t i i F th NEJM A hi C i ht © 2010 M h tt M di l S i t THE SEMINAL VESICLES IN GONORRHEA.1 BY CHARLES L. SCUDDER, M.D., BOSTON, Surgeon to the Massachusetts General Hospital Out-Patient Depart- ment; Assistant in Clinical and Operative Surgery, Harvard Uni- versity Medical School. BY CHARLES L. SCUDDER, M.D., BOSTON, SCUDDER, M.D., BOSTON, Surgeon to the Massachusetts General Hospital Out-Patient Depart- ment; Assistant in Clinical and Operative Surgery, Harvard Uni- versity Medical School. Seminal vesiculitis may be either acute or chronic. The occurrence of acute seminal vesiculitis during the course of an acute gonorrheal urethritis is rare. In 400 cases of gonorrhea, 160 of which cases were acute, there were 32 cases of prostatitis, 1 case of vesiculitis, and 15 cases of prostatitis and vesiculitis. These were cases observed by Colombini in 1897, and reported.2 Treatment of acute seminal vesiculitis. — At the outset, measures directed toward checking the inflam- matory process should be taken. If pus is present it should be evacuated either by incision and drainage through the rectum, or by incision and drainage through the perineum. Ch i i li i Ch i t oug p Chronic seminal vesiculitis.—Chronic vesiculitis is due either to indirect gonorrheal infection, resulting from an extension to the vesicle of inflammations con- nected with urethral lesions, the source of which was gonorrhea, or to tubercular infection. p Direct gonorrheal infection, that is, the extension from the urethra along the ejaculatorv duct and into the vesicle, is therefore extremely rare. Such in- flammations are very acute and of an extreme type. The contents of the vesicle become purulent, the vesicle wall and the perivesicular tissues are involved, and occasionally the peritoneum, which lies close to the summit of the vesicle, is also implicated. There are a few cases reported in which a general peritoni- tis has resulted from such peritoneal involvement. One vesicle alone is usually involved in the inflamma- tory process. i f i g , Symptoms of chronic seminal vesiculitis of gonor- rheal origin?— No inflammatory symptoms are pres- ent. The symptoms are functional or neurotic in character. Complaint is made of perverted sexual desire, irregular seminal emissions and neurotic sensa- tions. Certain mental symptoms may be present in these cases which are quite characteristic. q Many cases of so-called gleet may be found to be due to a chronic seminal vesiculitis, and many cases of chronic seminal vesiculitis may be the occasion of many of the neurotic and hypochondriacal symptoms seen in genito-urinary practice. y process. 3 Eugene Fuller : Disorders of the Male Sexual Organs. 4 Bolton: Journal of Genito-Urinary Diseases, vol. xvii, 1899, p. 651. THE SEMINAL VESICLES IN GONORRHEA.1 The element of germ infection in these cases is of very great importance. Beside the gonococcus in certain other cases presenting the signs of an acute seminal vesiculitis, the only germ found present has been the colon bacillus. It is probable that infection may take place, therefore, not only by continuity along the surface of the urethra through the seminal duct, but by direct invasion from contiguous tissues, such as the rectum. g y p The treatment by massage of the vesicles should be instituted before resorting to extirpation. Extirpa- tion of the sac of the seminal vesicle should be re- served for extreme cases, associated with serious or severe subjective symptoms. Th h j y p The operative routes to the seminal vesicles? — There are three routes by which the seminal vesicle may be reached, the inguinal, the perineal, or the sa- cral. The symptoms of acute seminal vesiculitis are al- most wholly inflammatory in character, characterized by fever, pain and tenderness. The pain may be re- ferred to the suprapubic region, to the sacrum, to the spermatic cord, to the scrotum, to the kidney, to the bladder, or to the urethra. The tenderness may be referred to the suprapubic region, where muscular rigidity may be evident, or to the region of the semi- nal vesicles by rectal palpation. The steps of the inguinal operation are as follows. The descriptions are those given by Bolton : (1) In- cision over the inguinal canal parallel with Poupart's ligament to scrotum ; (2) splitting aponeurosis of the external oblique over the canal, separation of vas, and enucleation of testis ; (3) ligation of deep epigastric vessels and incision of transversalis fascia ; (4) sepa- ration of peritoneum from iliac fossa wall and pelvis and bladder along the course of the vas deferens to the ureter ; (5) division of vas, its withdrawal from around the ureter ; (6) separation of vas and vesicle from base of bladder and from prostate and their re- moval. y p p The examination of the urine in these cases of acute vesiculitis is of the greatest importance. While purulent material is collecting within the sac of the vesicle, the acute symptoms are at their height. Im- mediately upon the emptying of the vesicle the acute symptoms subside, and at this time, when the symptoms have diminished in severity, pus appears in the urine. BY GARDNER W. ALLEN, M.D., BOSTON. eye p q The course of procedure in an average case of an- terior urethritis would be somewhat as follows : To begin with, when the meatus is very narrow it should be cut to a moderate degree, say to 26 or 28, as the So it appears that when gonorrhea has become localized in the deeper structures of the genito-uri- nary tract, energetic measures are called for. Time and perseverance are required. Sometimes little im- provement will be noticed, the discharge remaining about the same, being kept up perhaps by treatment. In such cases permanent improvement may be ex- pected after the active local measures have been brought to an end. And in cases of doubt as to whether this local treatment has been sufficiently thorough, it is well to suspend treatment for a time and note how much improvement follows and how permanent it may prove to be. So that while the acute inflammatory process is at its height the urine may be nearly clear, but it becomes purulent with the decrease of the symptoms and the escape of pus from the vesicular cavity. p p y The involvement of the perivesicular tissues is determined by palpation of the vesicles by rectum. The vesicles, instead of being easily detected in their swollen condition, are more or less obscured by a very great general swelling in their immediate neighbor- hood, and the edema or bogginess to the palpating finger extends over a wide area. When pus accumu- lates in these perivesicular tissues it may find an exit either into the peritoneal cavity or into the bladder, or into the rectum, or it may become absorbed. A few cases of pyemia have been reported which origi- nated in an acute seminal vesiculitis. Along with very acute seminal vesiculitis, the prostate is usu- ally somewhat enlarged, that is, congested, and press- ure upon the prostate will cause pain. The maxi- mum degree of tenderness is elicited when pressure is made over the seminal vesicle itself. The Boston Medical and Surgical Journal as published by The New England Journal of Medicine. Downloaded from nejm.org at UNIVERSITY COLLEGE LONDON on June 28, 2016. For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society The Boston Medical and Surgical Journal as published by y, p pp 1Read before the Boston Society for Medical Improvement, D cember 31, 1900. ».Centrbl. f. die Krankh. d. Ham. u. Sex. Org., 1897, S. 521. The Boston Medical and Surgical Journal as published by The New England Journal of Medicine. Downloaded from nejm.org at UNIVERSITY COLLEGE LONDON on June 28, 2016. For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. THE SEMINAL VESICLES IN GONORRHEA.1 y, p pp 1Read before the Boston Society for Medical Improvement, De- cember 31, 1900. ».Centrbl. f. die Krankh. d. Ham. u. Sex. Org., 1897, S. 521.
https://openalex.org/W2998584250	https://europepmc.org/articles/pmc6968335?pdf=render	English	null	Association between longer hospitalization and development of <i>de novo</i> donor specific antibodies in simultaneous liver–kidney transplant recipients	Renal failure	2,019	cc-by	6,536	CONTACT Miklos Z. Molnar mzmolnar@uthsc.edu Methodist Transplant Epidemiology Research Group, University of Tennessee Health Science Center, James D. Eason Transplant Institute, Methodist University Hospital, 1211 Union Avenue, Memphis, TN 38104, USA Supplemental data for this article can be accessed here. 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. 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 use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT Background: De novo Donor Specific Antibodies (DSA) are considered as a risk factor for the kid- ney allograft outcomes in recipients after simultaneous liver–kidney transplantation (SLKT). We hypothesized that length of hospital stay (LOS) might be associated with de novo DSA develop- ment of due to the increased likelihood of receiving blood transfusions with reduced immuno- suppressive regimens. KEYWORDS Donor specific antibody; DSA; de novo DSA; simultaneous liver–kidney transplantation; length of hospital stay; hospitalization pp g Methods: This study is a single-center, retrospective cohort study consisting of 85 recipients who underwent SLKT from 2009 to 2018 in our hospital. We divided the patients into two groups according to LOS [long hospital stay (L) group (LOS >14 days) and short hospital stay (S) group (LOS 14 days)]. Propensity score (PS) has been created using logistic regression to predict LOS greater than median of 14 days. The association between the presence of de novo DSA and LOS was assessed by logistic regression models adjusted for PS. y g g j Results: The mean age at transplantation of the entire cohort was 55.5 ± 10.1 years. Sixty percent of the recipients were male and Caucasian. Median LOS in (L) group was three-fold longer than (S) group [L: median 30 days (IQR: 21–52), S: median 8.5 days (IQR: 7–11)]. Eight patients devel- oped de novo DSA after SLKT (9.4%), all of them were in (L) group. Longer LOS was significantly associated with higher risk of development of de novo DSA in unadjusted (ORþ each 5 days: 1.09, 95% CI:1.02–1.16) and PS adjusted (ORþ each 5 days: 1.11, 95% CI:1.02–1.21) analysis. Conclusion: Longer hospitalization is significantly associated with the development of de novo ) j ( þ each 5 days , ) y Conclusion: Longer hospitalization is significantly associated with the development of de novo DSA in SLKT. CLINICAL STUDY Association between longer hospitalization and development of de novo donor specific antibodies in simultaneous liver–kidney transplant recipients Association between longer hospitalization and development of de novo donor specific antibodies in simultaneous liver–kidney transplant recipients Masahiko Yazawaa,b,c, Orsolya Cseprekala,b,d, Ryan A. Helmicka,b, Manish Talwara,b, Vasanthi Balaramana,b, Pradeep S. B. Podilae,f, Sallyanne Fosseyg, Sanjaya K. Satapathyh, James D. Easona,b and Miklos Z Molnara,b,d,i Masahiko Yazawaa,b,c, Orsolya Cseprekala,b,d, Ryan A. Helmicka,b, Manish Talwara,b, Vasanthi Balaramana,b, Pradeep S. B. Podilae,f, Sallyanne Fosseyg, Sanjaya K. Satapathyh, James D. Easona,b and Miklos Z. Molnara,b,d,i aJames D. Eason Transplant Institute, Methodist University Hospital, Memphis, TN, USA; bDivision of Transplant Surgery, Department of Surgery, University of Tennessee Health Science Center, Memphis, TN, USA; cDivision of Nephrology and Hypertension, Department of Internal Medicine, St. Marianna University School of Medicine, Kawasaki, Japan; dDepartment of Transplantation and Surgery, Semmelweis University, Budapest, Hungary; eFaith and Health Division, Methodist Le Bonheur Healthcare, Memphis, TN, USA; fDivision of Health Systems Management and Policy, School of Public Health, The University of Memphis, Memphis, TN, USA; gTransplant Immunology Laboratory, DCI Inc, Nashville, TN, USA; hSandra Atlas Bass Center for Liver Diseases and Transplantation, Department of Medicine, Northshore University Hospital/Northwell Health, Manhasset, NY, USA; iDivision of Nephrology, Department of Medicine, University of Tennessee Health Science Center, Memphis, TN, USA ARTICLE HISTORY Received 28 October 2019 Revised 3 December 2019 Accepted 4 December 2019 KEYWORDS Donor specific antibody; DSA; de novo DSA; simultaneous liver–kidney transplantation; length of hospital stay; hospitalization RENAL FAILURE 2020, VOL. 42, NO. 1, 40–47 https://doi.org/10.1080/0886022X.2019.1705338 RENAL FAILURE 2020, VOL. 42, NO. 1, 40–47 https://doi.org/10.1080/0886022X.2019.1705338 olnar@uthsc.edu Methodist Transplant Epidemiology Research Group, University of Tennessee Health Science tute, Methodist University Hospital, 1211 Union Avenue, Memphis, TN 38104, USA Introduction develop de novo DSA after LTA and SLKT [5,6,10]. Currently, the risk factors associated with newly devel- oped de novo DSA have not been well investigated in SLKT. The identification of potentially modifiable risk factors influencing de novo DSA development after SLKT might have positive effects on patient and graft survival. Post-transplant donor-specific antibodies (DSA), either identified pre-transplant (persistent DSA) or newly developed (de novo DSA) beyond the absorptive cap- acity conferred by allograft liver [1–4], present a risk fac- tor for patient- and allograft kidney outcome after simultaneous liver–kidney transplantation (SLKT) [5,6]. While the majority of pre-transplant DSA become undetectable after liver transplantation alone (LTA) [7] and after SLKT [8,9], about 10–20% of recipients Length of hospital stay (LOS) after surgery is one of the relevant clinical outcomes measured in many clin- ical settings [11–13]. Longer LOS has been shown to be RENAL FAILURE 41 to common statistical packages; and 4) procedures for importing data from external sources. associated with patient characteristics such as age, higher morbidity, worsened frailty, increased number and severity of comorbidities and unfavorable clinical outcomes and complications [11–16]. Previous studies also showed longer LOS was associated with more infectious complications; which could lead to decreased use of immunosuppressive medications or larger amount of blood product transfusions [14,15,17]. The clinical and research activities being reported are consistent with the Principles of the Declaration of Istanbul as outlined in the ‘Declaration of Istanbul on Organ Trafficking and Transplant Tourism’. All deceased donated organs were procured based on SLKT alloca- tion policy in Organ Procurement and Transplantation Network (OPTN)/United Network for Organ Sharing (UNOS) [25] and thus no organs were procured from prisoners [26]. Infectious complications and blood transfusions have also been identified as risk factors for longer LOS in liver transplant recipients [18–20]. Infectious complica- tions can cause cessation or reduction of immunosup- pressive medications; while blood transfusions can cause allo-sensitization [21,22]. Furthermore, early allo- graft liver dysfunction (EAD) was also identified as a risk factor for longer LOS [23]. EAD grafts may lose the cap- acity to fully absorb existing pre-transplant DSA, which might lead to persistent DSA after SLKT. Longer hospital stay might serve as a surrogate marker for these sensi- tization events, in addition to demonstrating associ- ation with de novo DSA development after SLKT. This study was approved by the Institutional Review Committee of The University of Tennessee Health Science Center (18-06146-XP). Introduction This is retrospective observational data collection and waiver for the con- sent form from participated recipients was approved by our IRB. Furthermore, this study did not need the con- sent from deceased donor either since this was not an interventional study [27]. Immunosuppression protocol In this retrospective study, we hypothesized that LOS is associated with a higher probability of de novo DSA development after SLKT. We evaluated the associ- ation between LOS and de novo DSA development using a single-center cohort in the modern immuno- suppressant era. The applied immunosuppression protocol was similar for all patients regardless of pre-transplant sensitization status [28]. As induction therapy, all patients received intravenous methylprednisolone (500 mg) on day 0, and rabbit anti-thymocyte globulin (1.5 mg/kg) on day 0 and again on post-operative day 2. Mycophenolate mofetil (MMF) or equivalent mycophenolic acid was started immediately post-operatively and continued until month three. Tacrolimus was started after improvement in kidney function, usually between post-operative day 3–7, and target trough range was 6–8 ng/mL until 3 months post-transplantation and 3–5 ng/mL thereafter. No patients received pre- transplant desensitization before SLKT. All patients were maintained on a steroid-free protocol. Cohort definition and data source This is a single-center, retrospective cohort study. We enrolled 85 consecutive recipients who underwent SLKT from 1 April 2009 to 28 February 2018 at Methodist University Hospital in Memphis, TN, USA. Exclusion criteria being those who were less than 18 years old or equal, but no patients were excluded from this study. Any information from recipients or deceased donors, as well as immunologic information were extracted from local electronic medical record (EMR), from the UNOS database, and from our HLA laboratory database until February 9th, 2019. We cap- tured all data into a Research Electronic Data Capture (REDCap) system, which is an electronic data capturing tool hosted at the Center for Biomedical Informatics, the University of Tennessee Health Science Center [24]. REDCap (Research Electronic Data Capture) is a secure, web-based application designed to support data cap- ture for research studies, providing: 1) an intuitive inter- face for validated data entry; 2) audit trails for tracking data manipulation and export procedures; 3) auto- mated export procedures for seamless data downloads 42 M. YAZAWA ET AL. 42 M. YAZAWA ET AL. Covariates than 1000. The median days of measurement of DSA from SLKT in both any post-transplant DSA and de novo DSA were also calculated as sum of days. The DSA measurements were performed as per clinical indica- tion. The methodology of detecting DSA including the decision of measurements, technical issues, and thresh- olds of DSA was not changed during the study period. We extracted data about recipients’ baseline character- istics including age, gender, race, body mass index (BMI), marital status, insurance, cause of end-stage liver disease (ESLD), cause of chronic kidney disease (CKD)/ end stage renal disease (ESRD), pre-SLKT dialysis infor- mation including length and type (maintenance dialysis was defined as dialysis for 3 months; acute dialysis initiation was defined as dialysis for <6 weeks; while sub-acute dialysis was defined as dialysis for 6 weeks- <3 months before transplantation), comorbid condi- tions (diabetes: DM and hypertension: HTN), the Model for End-stage Liver Disease (MELD) score at SLKT, the number of Human Leucocyte Antigen (HLA) mis- matches, calculated panel reactive antibody (cPRA), and cold-ischemic time (CIT) of donated kidney from the above mentioned sources. As post-SLKT information, first discharge destination, delayed allograft kidney function (DGF), primary non-function (PNF) on allograft kidney, death following transplant hospitalization. DGF was defined as needs of dialysis within one-week post SLKT [31] and PNF was defined as the condition on dia- lysis dependence after SLKT. Deceased donors’ informa- tion included age, sex, race, cause of death, history of hypertension and diabetes, and expanded criteria donor (ECD) status were also collected. All donations occurred after brain death. There was only one recipi- ent with missing value of CIT, thirteen recipients had missing value of cPRA, and one recipient had missing value of HLA mismatches. These missing values were not imputed. Statistical analysis Baseline characteristics were described for the entire cohort and for groups categorized based on the LOS and presented as mean ± standard deviation (SD) or median and interquartile range (IQR) for continuous variables and percent for categorical variables as appro- priate. Differences between groups were assessed by student T-test or Mann-Whitney test for continuous var- iables and chi-square-test (or Fisher’s exact test) for cat- egorical variables. We calculated the propensity score (PS) for probabil- ity of long hospital stay in the main analysis (LOS >14 days versus 14 days) and in the sensitivity ana- lysis (LOS >28 days versus 28 days) using logistic regression model (presented in Table S1, Supplementary material), including all available covari- ates without missing values for LOS, including age, gen- der, race, marital status, insurance, cause of ESLD, diabetes, hypertension, BMI, blood type, MELD score, first discharge destination, and DGF. The purpose of this step was to be able to adjust for co-variates which showed association with LOS. Because only 8 patients developed de novo DSA, we were able to adjust for only one variable in our adjusted logistic regression analysis used for assessing association between LOS (as exposure) and development of de novo DSA or any post-transplant DSA (as outcomes). We then assessed logistic regression analysis, unadjusted and PS score adjusted, to calculate the relative risk (odds ratio) for newly developing de novo DSA or any post-trans- plant DSA. Outcomes Primary outcome was incidence of development of de novo DSA after SLKT. Furthermore, prevalence of post- transplant DSA (persistent or de novo DSA), was also assessed as secondary outcome. Persistent DSA was defined as DSA detected before and after SLKT, while de novo DSA was defined as newly developed DSA fol- lowing SLKT. We assessed the incidence of persistent DSA or de novo DSA or in each patient after SLKT. HLA specificities were identified using a solid phase single antigen bead platform (SAB; One Lambda Inc, a division of Thermo-Fisher, Canoga Park, CA, USA) combined with Luminex xMAP technology (Luminex Corporation., Northbrook, IL, USA). Patients with any observed class DSA were categorized as de novo DSA (þ), while those negative for identified de novo DSA were classified de novo DSA (). We defined post-DSA that has a mean fluorescence intensity (MFI) value that is elevated com- pared to pre-transplant levels or has an MFI of greater p values were two-sided and significance level was set at less than .05 for all analyses. All analyses were conducted using STATA Version 13 (STATA Corporation, College Station, TX). Exposure The LOS was defined as the exposure in this study. LOS was calculated as sum of the days from date of admis- sion to date of discharge. LOS was divided into two groups according to the median of 14 days. Long hos- pital stay group was defined as LOS greater than 14 days [(L) group] and short hospital stay group was defined as LOS less than 14 days or equal [(S) group]. For the sensitivity analysis, the threshold of long LOS was replaced with 28 days, which was two-times longer compared to the main analysis and based on the previ- ous literature [29,30]. Baseline characteristics Table 1 shows the baseline characteristics of the entire cohort for both (L) and (S) groups. The median age at SLKT was 55.5 ± 10.1 years old and approximately 60% of the patients were Caucasian males. The leading RENAL FAILURE 43 le 1. Baseline characteristics of the entire cohort and divided by long and short hospital stay groups. stics of the entire cohort and divided by long and short hospital stay groups. Table 1. Baseline characteristics of the entire cohort and divided by long and short hospital stay groups. Baseline characteristics Baseline characteristics Entire cohort, N ¼ 85 Long hospital stay group (L), N ¼ 43 Short hospital stay group (S), N ¼ 42 p Value Recipient information Age, years, mean ± SD 55.5 ± 10.1 56.7 ± 9.4 54.4 ± 10.7 .292 Gender, male, n (%) 53 (62.4) 28 (65.1) 25 (59.5) .595 BMI, kg/m2, mean ± SD 27.0 ± 6.4 28.2 ± 7.2 28.6 ± 5.6 .827 Race, n (%) .866 African American 22 (25.9) 11 (25.6) 11 (26.2) Caucasian 51 (60.0) 26 (60.5) 25 (59.5) Other 12 (14.1) 6 (14.0) 6 (14.3) Marital status, Married, n (%) 52 (61.2) 27 (62.8) 25 (59.5) .783 Insurance, n (%) .680 Private 31 (36.5) 16 (37.2) 15 (35.7) Medicaid 6 (7.1) 4 (9.3) 2 (4.8) Medicare 48 (56.5) 23 (53.5) 25 (59.5) Presence of preexisting CKD, n (%) 74 (87.1) 32 (74.4) 42 (100) <.001 Cause of CKD, n (%) .647 Hypertension 8/74 (10.8) 4/32 (12.5) 4/42 (9.5) Diabetes 14/74 (18.9) 6/32 (18.8) 8/42 (19.0) Glomerulonephritis 4/74 (5.4) 2/32 (6.3) 2/42 (4.8) Cystic disease 3/74 (4.1) 0 3/42 (7.1) Metabolic/inherited disease 2/74 (2.7) 0 2/42 (4.8) Other/unknown 43/74 (58.1) 20/32 (62.5) 23/42 (54.8) Dialysis status before SLKT, n (%) .003 Maintenance dialysis, n (%) 38 (44.7) 19 (44.2) 19 (45.2) Sub-acute dialysis, n (%) 9 (10.6) 6 (14.0) 3 (7.1) Acute dialysis initiation before SLKT, n (%) 16 (18.8) 13 (30.2) 3 (7.1) Length of dialysis before SLKT (maintenance and sub-acute dialysis group), months, median (IQR) 8.9 (3.9, 36.8) 7.1 (3.3, 20.4) 13.0 (7.4, 44.6) .077 Length of acute dialysis before SLKT (acute dialysis group only), days, median (IQR) 13.5 (6.5, 24.5) 12.0 (8.0, 24.0) 19.0 (5.0, 27.0) .638 Cause of ESKD (maintenance and sub-acute group), n (%) .278 Acute on CKD, 12/47 (25.5) 8/25 (32.0) 4/22 (18.2) Same as CKD 35/47 (74.5) 17/25 (68.0) 18/22 (81.8) Cause of ESLD, n (%) .127 HCV 24 (28.2) 7 (16.3) 17 (40.5) Alcoholic hepatitis 22 (25.9) 14 (32.6) 8 (19.0) HCV and Alcoholic hepatitis 3 (3.5) 1 (2.3) 2 (4.8) NASH 16 (18.8) 9 (20.9) 7 (16.7) Other 20 (23.5) 12 (27.9) 8 (19.0) Comorbidity – diabetes, n (%) 35 (41.2) 18 (41.9) 17 (40.5) .897 Comorbidity – hypertension, n (%) 61 (71.8) 29 (67.4) 32 (76.2) .370 HLA mismatches locus A, n, mean ± SD 1.6 ± 0.6 1.5 ± 0.7 1.5 ± 0.6 .859 HLA mismatches locus B, n, mean ± SD 1.7 ± 0.5 1.7 ± 0.5 1.7 ± 0.5 .820 HLA mismatches locus DR, n, mean ± SD 1.5 ± 0.5 1.5 ± 0.6 1.6 ± 0.5 .535 Total HLA mismatches, n, mean ± SD 4.8 ± 1.0 4.8 ± 1.1 4.9 ± 0.9 .750 cPRA, %, median (IQR) 0 (0, 8) 0 (0, 3.0) 0 (0, 13.0) .866 Cold ischemic time of donated kidney, minutes, mean ± SD 496.5 ± 114.6 514.3 ± 114.1 478.6 ± 113.6 .155 MELD score, mean ± SD 28.3 ± 6.5 31.7 ± 6.3 24.9 ± 4.6 <.001 Donor information Age, years, mean ± SD 28.9 ± 11.1 29.2 ± 10.9 28.6 ± 11.4 .793 Gender, male, n (%) 47 (55.3) 27 (62.8) 20 (47.6) .160 Donor Race, n (%) .163 Caucasian 65 (76.5) 36 (83.7) 29 (69.0) African American 18 (21.2) 7 (16.3) 11 (26.2) Hispanic 2 (2.4) 0 2 (4.8 Donation after brain death, n (%) 85 (100) 43 (100) 42 (100) – Cause of death, n (%) .785 Anoxia 27 (31.8) 14 (32.6) 13 (31.0) Cerebrovascular/stroke 18 (21.2) 8 (18.6) 10 (23.8) Head trauma 33 (38.8) 18 (41.9) 15 (35.7) Central nerve system tumor 1 (1.2) 0 1 (2.4) Other 5 (5.9) 2 (4.7) 3 (7.1) Comorbidity-diabetes, n (%) 1 (1.2) 1 (2.3) 0 .320 Comorbidity-hypertension, n (%) 11 (12.9) 7 (16.3) 4 (9.5) .354 Expanded criteria donor, n (%) 1 (1.2) 1 (2.3) 0 .320 BMI: Body mass index; CKD: Chronic kidney disease; cPRA: calculated panel reactive antibody; ESKD: End-stage kidney disease; ESLD: End-stage liver dis- ease; HCV: Hepatitis C; HLA: Human Leukocyte Antigen; IQR: Interquartile range; MELD: Model of end-stage liver disease; NASH: Nonalcoholic steatohepati- tis; SLKT: Simultaneous liver–kidney transplantation. BMI: Body mass index; CKD: Chronic kidney disease; cPRA: calculated panel reactive antibody; ESKD: End-stage kidney disease; ESLD: End-stage liver dis- ease; HCV: Hepatitis C; HLA: Human Leukocyte Antigen; IQR: Interquartile range; MELD: Model of end-stage liver disease; NASH: Nonalcoholic steatohepati- tis; SLKT: Simultaneous liver–kidney transplantation. Compared between Long hospital stay group (L) group and short hospital stay group (L) groups. p values for continuous variables with mean ± SD are result of t-test and with median (IQR) are result of Mann–Whitney test, and categorical variables are chi-square test. Baseline characteristics Compared between Long hospital stay group (L) group and short hospital stay group (L) groups. p values for continuous variables with mean ± SD are result of t-test and with median (IQR) are result of Mann–Whitney test, and categorical variables are chi-square test. Comorbidity – diabetes, n (%) Comorbidity – hypertension, n (%) HLA mismatches locus B, n, mean ± SD BMI: Body mass index; CKD: Chronic kidney disease; cPRA: calculated panel reactive antibody; ESKD: End-stage kidney disease; ESLD: End-stage liver dis- ease; HCV: Hepatitis C; HLA: Human Leukocyte Antigen; IQR: Interquartile range; MELD: Model of end-stage liver disease; NASH: Nonalcoholic steatohepati- tis; SLKT: Simultaneous liver–kidney transplantation. Compared between Long hospital stay group (L) group and short hospital stay group (L) groups. p values for continuous variables with mean ± SD are result of t-test and with median (IQR) are result of Mann–Whitney test, and categorical variables are chi-square test. 44 M. YAZAWA ET AL. . Post-transplant characteristics of the entire cohort and divided by long and short hospital stay groups. Table 2. Post-transplant characteristics of the entire cohort and divided by long and short hos Table 2. Post-transplant characteristics of the entire cohort and divided by long and short hospital stay groups. Baseline characteristics Entire cohort, N ¼ 85 Long hospital stay group, (L) N ¼ 43 Short hospital stay group (S), N ¼ 42 p Value Length of stay for SLKT admission, days, me\dian (IQR) 15.0 (9.0, 30.0) 30.0 (21.0, 52.0) 8.5 (7.0, 11.0) <.001 Duration from admission to transplantation, days, median (IQR) 1.0 (1.0, 6.0) 6.0 (1.0, 16.0) 0 (0, 1.0) <.001 Duration from transplantation to discharge, days, median (IQR) 11.0 (8.0, 21.0) 21.0 (12.0, 42.0) 8.0 (6.0, 10.0) <.001 Delayed graft function (kidney), n (%) 15 (17.6) 12 (27.9) 3 (7.1) .012 Primary non-function (kidney), n (%) 0 0 0 – Death during hospitalization, n (%) 9 (10.6) 7 (16.3) 2 (4.8) .084 First discharge destination, n (%) .001 Home 41 (48.2) 12 (27.9) 29 (69.0) Home and taking health service 22 (25.9) 13 (30.2) 9 (21.4) Rehabilitation hospital 12 (14.1) 10 (23.3) 2 (4.8) Others 10 (11.8) 8 (18.6) 2 (4.8) Incidence of developing of de novo DSA, n (%) 8 (9.4) 8 (18.6) 0 .003 Duration from transplant to measurement of de novo DSA, days, median (IQR) 53.0 (14.0, 280.5) 53.0 (14.0, 280.5) N/A – Prevalence of any post-transplant DSA, n (%) 12 (14.1) 11 (25.6) 1 (2.4) .002 DSA: Donor specific antibody; IQR: Interquartile range; SLKT: Simultaneous liver–kidney transplantation. Compared between long hospital stay and short hospital stay groups. p values for continuous variables with median (IQR) are result of Mann–Whitney test and categorical variables are chi-square test. tics of the entire cohort and divided by long and short hospital stay groups. DSA: Donor specific antibody; IQR: Interquartile range; SLKT: Simultaneous liver–kidney transplantation. Compared between long hospital stay and short hospital stay groups. p values for continuous variables with median (IQR) are result of Mann–Whitney test and categorical variables are chi-square test. Figure 1. Flow chart of patient selection and incidence and prevalence of de novo DSA and persistent DSA. Abbreviations: DSA: Donor-specific antibody; de novo DSA: newly developed DSA; N: Number; SLKT: Simultaneous liver–kidney transplantation. causes of ESLD were hepatitis C and alcoholic hepatitis. The patients in (L) group had significantly higher MELD scores, prevalence of acute dialysis initiation shorter length of dialysis before SLKT (maintenance and sub- acute dialysis) and higher prevalence of alcoholic hepa- titis as a cause of ESLD compared with (S) group patients. Length of hospital stay and post-transplant events Table 2 shows post-SLKT information of LOS, first dis- charge destination, and incidence of outcomes, DGF, PNF, and death following SLKT hospitalization. The median LOS of entire cohort was 15 days (IQR: 9–30 days) and duration from transplantation to dis- charge was median 11 days (IQR: 8–21 days). The median LOS in (L) group was significantly longer than that in (S) group [30 days (IQR: 21–52 days) and 8.5 days (IQR: 7–11 days), p < .001], respectively. In (L) group, incidence of returning to home as first discharge destin- ation was significantly lower than in (S) group (27.9% and 69.0%, respectively, p ¼ .001). No PNF was observed; however, DGF of the allograft kidney was sig- nificantly higher in (L) group. Incidence of death during SLKT hospitalization was higher in (L) group compared to (S) group. Figure 1. Flow chart of patient selection and incidence and prevalence of de novo DSA and persistent DSA. Abbreviations: DSA: Donor-specific antibody; de novo DSA: newly developed DSA; N: Number; SLKT: Simultaneous liver–kidney transplantation. Developing of de novo DSA and prevalence of any post-transplant DSA Developing of de novo DSA and prevalence of any post-transplant DSA Developing of de novo DSA and prevalence of any post-transplant DSA transplant DSA were in (L) group. The median days from SLKT was 22.0 days (IQR: 8.0–280.5 days) for meas- urement of any post-transplant DSA and 53.0 days was (IQR: 14.0–280.5 days) for measurement of de novo DSA (Table 2). Eight patients developed de novo DSA (9.4%) and 12 patients were identified with any post-transplant DSA consisted of both persistent and de novo DSA (14.1%) after SLKT (Figure 1). All patients who developed de novo DSA and 11 out of 12 patients with any post- RENAL FAILURE 45 45 45 Table 3. Probability of development of de-novo DSA and any post-transplant DSA using unadjusted and propensity score adjusted logistic regression models. Table 3. Probability of development of de-novo DSA and any post-transplant DSA using unadjusted and propensity score adjusted logistic regression models. Table 3. Probability of development of de-novo DSA and any post-transplant DSA using unadjusted and propensity score adjusted logistic regression models. Risk of de novo DSA Risk of any post-transplant DSA Odds ratio 95% CI p value Odds ratio 95% CI p value Un-adjusted Length of hospital stay (each 5 days) 1.09 1.02–1.16 .010 1.08 1.02–1.15 .011 Adjusted Length of hospital stay (each 5 days) 1.11 1.02–1.21 .014 1.08 1.01–1.16 .028 PS score 0.27 0.02–4.13 .343 1.08 0.13–8.68 .944 DSA: Donor specific antibody; PS: Propensity score; 95% CI: 95% confidence interval. Sensitized status in SLKT has tended to be neglected due to the absorptive capacity by allograft liver [1–4]. In fact, sensitization before SLKT is not a contraindication of transplantation from several clinical practice guide- lines [32–34]. However, post-transplant DSA, especially de novo ClassII DSA, has been thought to be a signifi- cant risk factor for patient-, allograft liver-, and allograft kidney outcome [5]. No previous study has assessed the risk factors of developing de novo DSA in SLKT, although the potential for exposure to sensitizing events might be expected to be higher in SLKT than liver or kidney transplantation alone (LTA and KTA). Although LTA patients typically do not undergo main- tenance dialysis therapy before LTA, 30–50% of SLKT candidates undergo dialysis therapy immediately prior to SLKT [35,36]. We observed this finding in our cohort as well. Simultaneous liver–kidney transplant patients could be more fragile prior to transplantation and require more blood transfusions peri-operatively com- pared to KTA patients [37]. Probability of developing de novo DSA and any post-transplant DSA in the sensitivity analysis Probability of developing de novo DSA and any post-transplant DSA in the sensitivity analysis Longer LOS defined as >28 days was significantly asso- ciated with higher risk of development of de novo DSA and prevalence of any post-transplant DSA consisted of both persistent and de novo DSA in unadjusted and PS adjusted analysis (Table S2, Supplementary material). Probability of developing de novo DSA and any post-transplant DSA Longer LOS was significantly associated with higher risk of development of de novo DSA in unadjusted (ORþ each 5 days: 1.09, 95% CI: 1.02–1.16) and PS adjusted (ORþ each 5 days: 1.11, 95% CI: 1.02–1.21) analysis (Table 3). Longer LOS was also significantly associated with higher risk of prevalence of any post-transplant DSA consisted of both persistent and de novo DSA in unadjusted (ORþ each 5 days: 1.08, 95% CI: 1.02–1.15) and PS adjusted (ORþ each 5 days: 1.08, 95% CI: 1.01–1.16) ana- lysis (Table 3). Developing of de novo DSA and prevalence of any post-transplant DSA Higher prevalence of main- tenance dialysis therapy and history of blood transfusions in SLKT patients has been associated with sensitization [38–40], which leads to higher probability of sensitization before and after transplantation com- pared to LTA and KTA patients. Probability of developing de novo DSA and any post-transplant DSA References [1] Zavazava N. Soluble HLA class I molecules: biological significance and clinical implications. Mol Med Today. 1998;4:116–121. [1] Zavazava N. Soluble HLA class I molecules: biological significance and clinical implications. Mol Med Today. 1998;4:116–121. [2] Gugenheim J, Amorosa L, Gigou M, et al. Specific absorption of lymphocytotoxic alloantibodies by the liver in inbred rats. Transplantation. 1990;50:309–313. [2] Gugenheim J, Amorosa L, Gigou M, et al. Specific absorption of lymphocytotoxic alloantibodies by the liver in inbred rats. Transplantation. 1990;50:309–313. Despite some limitations, our study has confirmed several previous findings. LOS has been shown to be predicted by factors such as ethnicity, discharge destin- ation, and type of insurance [13]. In our study, (L) group patients were more likely to be discharged to rehabilita- tion or discharged with home service than (S) group. Despite have low event numbers, we were able to adjust for these variables using a PS score. This is the first report to identify LOS as a risk factor for de novo DSA development in SLKT. Our results support the con- clusion that DSA monitoring can be implemented into clinical practice for SLKT patients. [3] Taner T, Park WD, Stegall MD. Unique molecular changes in kidney allografts after simultaneous liver- kidney compared with solitary kidney transplantation. Kidney Int. 2017;91:1193–1202. [3] Taner T, Park WD, Stegall MD. Unique molecular changes in kidney allografts after simultaneous liver- kidney compared with solitary kidney transplantation. Kidney Int. 2017;91:1193–1202. [4] Taner T, Gustafson MP, Hansen MJ, et al. Donor-spe- cific hypo-responsiveness occurs in simultaneous liver- kidney transplant recipients after the first year. Kidney Int. 2018;93:1465–1474. [4] Taner T, Gustafson MP, Hansen MJ, et al. Donor-spe- cific hypo-responsiveness occurs in simultaneous liver- kidney transplant recipients after the first year. Kidney Int. 2018;93:1465–1474. [5] O’Leary JG, Gebel HM, Ruiz R, et al. Class II alloanti- body and mortality in simultaneous liver-kidney trans- plantation. Am J Transplant. 2013;13:954–960. [6] [6] Yazawa M, Cseprekal O, Helmick AR, et al. Association between post-transplant donor specific antibodies and recipient outcomes in simultaneous liver-kidney transplant recipients: single center, cohort study. Transpl Int. 2019; doi:10.1111/tri.13543. [Epub ahead of print] PubMed PMID: 31647143. In conclusion, longer hospitalization was significantly associated with higher probability of persistent DSA and de novo DSA development after SLKT. The authors report no conflict of interest. The authors report no conflict of interest. The authors report no conflict of interest. Acknowledgments The results of this paper have not been published previously in whole or part. M. YAZAWA ET AL. 46 and larger studies are highly warranted to identify more modifiable and non-modifiable predictors for per- sistent and de novo DSA development following SLKT. Longer hospitalization also showed significant asso- ciation with a higher probability of persistent or de novo post-transplant DSA. In our cohort, 4 patients had persistent DSA; 2 patients had both persistent and de novo DSA after SLKT (Figure 1). Five of the six patients with persistent DSA after SLKT, were in (L) group. Our results suggest patients with pre-transplant DSA and longer LOS should be routinely monitored for post- transplant DSA. Data availability statement The datasets generated during the current study are avail- able from the corresponding author on reasonable request. Author contributions Several limitations should be noted with this study. Although this is one of the largest SLKT cohorts studied to date, our patient group was still small and had a rela- tively low number of events. Because this was a retro- spective cohort study, we could not conclude causal relationship between LOS and developing de novo DSA despite the finding the median days of measurement of DSA after SLKT was almost double compared to length of hospital stay in (L) group. Furthermore, DSA meas- urement was indication based and not routine, which might have caused observational bias. In this study, we were not able to assess the pathophysiological role of Class I and II DSA separately, as almost all of the patients with post-transplant DSA had at least Class II DSA (N ¼ 11/12) (Figure 1). Finally, the observed differ- ences in clinical practices observed across different transplant programs may lead to different definitions of LOS. These differences could be the generalizability of our findings. In fact, albeit LOS was replaced with 28 days or longer in our sensitivity analysis, LOS was still significant risk factor for the developing of de novo DSA and prevalence of any post-transplant DSA. Additional prospective and larger studies that include protocol DSA measurement are highly warranted. All authors contributed to conception and design of the study and approved the final version of the manuscript. M.Y., O.C., P.S.B.P., and M.Z.M. collected the clinical data. S.F exam- ined all donor specific antibodies. M.Y. and M.Z.M. performed the analysis and contributed to interpretation of results. M.Y., and M.Z.M. wrote the manuscript. M.Z.M. takes responsibility for the data and analysis accuracy and all other aspect the work. Discussion In this single center, retrospective study, we found sig- nificant associations between longer hospitalization and higher probability of both persistent post-trans- plant DSA and de novo DSA development after SLKT. In addition, our study indicates that longer LOS occurred more frequently in SLKT patients with higher MELD scores and higher incidence of DGF in allograft kidney. Furthermore, 30% of the patients in (L) group, com- pared to exact percentage in (S) group, were dis- charged to their home after SLKT. All of those who developed de novo DSA had longer hospitalizations. Our study implies that LOS might be a useful surrogate marker of a higher probability for persistent DSA and de novo DSA development. Patients, if not all, with lon- ger hospitalization should be routinely screened for DSA after SLKT. We believe this is the first report to evaluate an association between the length of hospital- ization and post-transplant DSA development in SLKT. We constructed a conceptual model of the assessed relationship between LOS and DSA. We identified an association between longer LOS and developing de novo DSA. The relationship between assumptive expo- sures, (blood transfusions, infectious events) and EAD and LOS has already been reported in other patient cohorts [14,15] as well as recipients with LTA [18–20,23]. We wanted to clarify a direct relationship between presumptive exposures and de novo DSA development. Our preliminary findings suggest LOS might be a consequence of DGF on allograft kidney secondary to persistent DSA or de novo DSA develop- ment or other potential intermediated mediators (indi- cator). Although we could not identify the exposure in each patient, a relationship between presumptive expo- sures and de novo DSA development could exist in accordance with this conceptual model. 46 M. YAZAWA ET AL. References [15] Micek S, Johnson MT, Reichley R, et al. An institutional perspective on the impact of recent antibiotic expos- ure on length of stay and hospital costs for patients with gram-negative sepsis. BMC Infect Dis. 2012;12:56. [31] Siedlecki A, Irish W, Brennan DC. Delayed graft func- tion in the kidney transplant. Am J Transplant. 2011; 11:2279–2296. [32] Davis CL, Feng S, Sung R, et al. Simultaneous liver-kid- ney transplantation: evaluation to decision making. Am J Transplant. 2007;7:1702–1709. [16] Baek H, Cho M, Kim S, et al. Analysis of length of hos- pital stay using electronic health records: a statistical and data mining approach. PLoS One. 2018;13: e0195901. [33] Eason JD, Gonwa TA, Davis CL, et al. Proceedings of con- sensus conference on simultaneous liver kidney trans- plantation (SLK). Am J Transplant. 2008;8:2243–2251. [17] Arefian H, Hagel S, Heublein S, et al. Extra length of stay and costs because of health care-associated infec- tions at a German university hospital. Am J Infect Control. 2016;44:160–166. [34] Hussain SM, Sureshkumar KK. Refining the role of sim- ultaneous liver kidney transplantation. J Clin Transl Hepatol. 2018;6:289–295. [18] Singh N, Paterson DL, Gayowski T, et al. Predicting bacteremia and bacteremic mortality in liver trans- plant recipients. Liver Transpl. 2000;6:54–61. [35] Locke JE, Warren DS, Singer AL, et al. Declining out- comes in simultaneous liver-kidney transplantation in the MELD era: ineffective usage of renal allografts. Transplantation. 2008;85:935–942. 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https://openalex.org/W2972512710	http://jurnal.borneo.ac.id/index.php/borneo_humaniora/article/download/839/561	Indonesian	null	DESKRIPSI HUBUNGAN MASYARAKAT DENGAN SEKOLAH DI SDIT ULUL ALBAB TARAKAN	Jurnal Borneo Humaniora	2,019	cc-by-sa	4,156	DESKRIPSI HUBUNGAN MASYARAKAT DENGAN SEKOLAH DI SDIT ULUL A Donna Rhamdan1, Jumriani2, Diana Perminas3, Satriani4 Program Studi Pendidikan Guru Sekolah Dasar Fakultas Keguruan Dan Ilmu Pendidikan Universitas Borneo Tarakan Email: donna.rhamdan@borneo.ac.id1 Donna Rhamdan , Jumriani , Diana Perminas , S Program Studi Pendidikan Guru Sekolah Dasar Fakultas Keguruan Dan Ilmu Pendidikan Universitas Borneo Tarakan Email: donna.rhamdan@borneo.ac.id1 ABSTRACT This study aims to determine the public relations with school at SDIT Ulul Albab Tarakan. This study uses a type of qualitative research with a descriptive approach. Data collection techniques in this study through observation, interviews, and documentation studies. The results showed that in the school had done an appropriate way to get support and sympathy from the public, by providing real information through social media, so that the role of public relations at SDIT Ulul Albab Tarakan was to good cooperation. The purpose of public relations with schools is to get support from the publics, and School has received support from the public in the form of a good positive response and public trust in SDIT Ulul Albab Tarakan. The form of public relations implementation is a function of public relations with schools, namely to overcome the problem of public relations with schools to achieve common goals and the has gone well so that the publics no one complains and the publics accepts well. The benefits of the form of public relations collaboration with schools have been going well and the form or technique in the implementation of work programs from public relations continuously. ABSTRAK Penelitian ini mengenai manajemen hubungan masyarakat dengan sekolah di SDIT Ulul Albab Tarakan. Pendekatan penelitian dengan kualitatif deskriptif. Teknik pengumpulan data yang digunakan melalui observasi, wawancara, dan studi dokumentasi. Hasil penelitian menunjukkan bahwa sekolah ini telah melakukan cara yang sesuai untuk mendapatkan dukungan dan simpati dari masyarakat, yaitu dengan memberikan informasi yang nyata melalui media sosial, sehingga peran humas di SDIT Ulul Albab Tarakan yaitu menjaga hubungan yang baik . Tujuan hubungan masyarakat dengan sekolah adalah mendapatkan dukungan dari masyarakat dan Sekolah telah mendapatkan dukungan dari masyarakat berupa respon positif yang baik dan kepercayaan masyarakat kepada Sekolah ini. Bentuk penyelenggaraan humas merupakan fungsi dari humas dengan sekolah yaitu untuk mengatasi persoalan hubungan masyarakat dengan sekolah. Di sekolah ini sudah berjalan dengan baik fungsi humas sehingga masyarakat tidak keberatan dan tidak ada yang komplain serta masyarakat menerima dengan baik. Manfaat dari bentuk kerjasama humas dengan sekolah sudah berjalan dengan baik serta bentuk atau teknik dalam pelaksanaan program kerja dari humas berkesinambungan. Donna Rhamdan, Jumriani, Diana Perminas, Satriani DESKRIPSI HUBUNGAN MASYARAKAT… Available online at jurnal.borneo.ac.id Diterbitkan Agustus 2019 Halaman 01-07 Available online at jurnal.borneo.ac.id Diterbitkan Agustus 2019 Halaman 01-07 Available online at jurnal.borneo.ac.id Diterbitkan Agustus 2019 Halaman 01-07 JURNAL BORNEO HUMANIORA e-ISSN 2599-3305 p-ISSN 2615-4331 http:// jurnal.borneo.ac.id/index.php/borneo_humaniora 2. Metode Pengumpulan Data Peran hubungan masyarakat sangat penting bagi sebuah organisasi atau lembaga termasuk hubungan masyarakat dengan sekolah. Karena pendidikan merupakan tanggung jawab stakeholder, maka diperlukan keterbukaan. Perencanaan kegiatan sekolah perlu disampaikan dengan baik kepada masyarakat, agar masyarakat dapat memahami dan memberikan dukungan. Oleh sebab itu setiap sekolah harus memiliki manajemen humas yang berfungsi untuk menyampaikan segala informasi kepada masyarakat yang ada diluar sekolah. Data penelitian diperoleh berdasarkan sumber data dengan menggunakan teknik pengumpulan data. Pengumpulan data dapat dilakukan melalui berbagai sumber dan berbagai cara. Menggunakan teknik observasi wawancara, dan studi dokumentasi. Adapun penjelasan mengenai teknik pengumpulan data tersebut dapat dilihat dibawah ini: PENDAHULUAN suatu negara karena suatu bangsa dikenal dan dipandang berdasarkan karakteristik dari bangsanya. Pendidikan tidak hanya berfokus kepada pembangunan aspek kognitif (pengetahuan) saja, namun juga memperhatikan perkembangan aspek afektif (sikap) dan psikomotorik (keterampilan) sehingga diharapkan nantinya Pendidikan merupakan pondasi bagi suatu negara agar dapat menjadi negara maju. Melalui pendidikan dapat dihasilkan generasi-generasi penerus bangsa yang dapat membawa perkembangan bagi suatu negara. Pendidikan juga mempengaruhi pandangan suatu bangsa terhadap Page 1 Available online at jurnal.borneo.ac.id Diterbitkan Agustus 2019 Halaman 01-07 JURNAL BORNEO HUMANIORA e-ISSN 2599-3305 p-ISSN 2615-4331 http://jurnal.borneo.ac.id/index.php/borneo_humaniora JURNAL BORNEO HUMANIORA e-ISSN 2599-3305 p-ISSN 2615-4331 http://jurnal.borneo.ac.id/index.php/borneo_humaniora Diterbitkan Agustus 2019 Halaman 01-07 seorang individu yang cerdas secara intelektual, memiliki tata krama yang baik, serta memiliki keterampilan yang dapat diandalkan. Informasi yang diberikan dapat berupa program-program sekolah maupun masalah- masalah yang sedang dihadapi sekolah dalam upaya peningkatan kualitas sekolah. Hal ini bertujuan agar masyarakat dapat mengetahui informasi yang ada di sekolah dan dapat dimengerti oleh pihak-pihak lain. Oleh karena itu setiap sekolah harus memiliki manajemen humas yang berfungsi untuk menyampaikan informasi kepada masyarakat. Pelaksanaan pendidikan tentunya tidak lepas dari adanya kegiatan pembelajaran yang menunjang jalannya pendidikan. Sebagaimana yang kita ketahui pendidikan merupakan wadah yang mewadahi terjadinya proses belajar mengajar atau yang disebut dengan kegiatan pembelajaran. Kegiatan pembelajaran merupakan aktivitas sehari- hari yang terjadi di dunia pendidikan khususnya di lingkungan sekolah yang merupakan salah satu lembaga pendidikan. Sekolah dirancang untuk memberikan pengajaran kepada peserta didik. Dalam memberikan pengajaran kepada peserta didik, sekolah perlu meningkatkan mutu pendidikan, salah satunya dengan adanya program pemerintah yang memberikan kewenangan kepada pihak sekolah. Manajemen humas dengan sekolah merupakan segala rangkaian kegiatan sekolah dengan masyarakat bertujuan menunjang pembelajaran di sekolah. Dalam penelitian ini membahas mengenai manajemen hubungan sekolah dengan masyarakat. Adapun penelitian bertujuan mengetahui hubungan masyarakat dengan SDIT Ulul Albab Tarakan. 1. Lokasi Penelitian Sesuai dengan UU No. 20 Tahun 2003 tentang SISDIKNAS pasal 51 ayat 1 yang menyatakan bahwa manajemen berbasis sekolah atau madrasah adalah bentuk otonomi manajemen pendidikan pada satuan pendidikan, yang dalam hal ini kepala sekolah atau madrasah dan guru dibantu oleh komite sekolah atau madrasah dalam mengelola kegiatan pendidikan. Untuk menjalankan kegiatan tersebut diperlukan adanya kerjasama antara semua stakeholder yang akan mempermudah meningkatkan kualitas sekolah. 1. Lokasi Penelitian Peneliti memilih dan menempatkan lokasi penelitian di sekolah berprestasi. Pemilihan ini didasarkan dengan alasan untuk mendapat informasi mengenai objek materi yang diteliti dan data yang sesuai dengan fokus penelitian. Setelah dilakukan beberapa pertimbangan, sekolah yang dijadikan subjek penelitian yaitu SDIT Ulul Albab Tarakan. Adapun waktu penelitian pada tanggal 4 Desember 2018. d. Prosedur Penelitian Untuk memperoleh hasil analisis data, peneliti melakukan tahap-tahap sebagai berikut: Dalam penelitian yang pertama kali dilakukan adalah melakukan observasi, dicatat maupun dijadikan data umum. Membuat pertanyaan tentang permasalahan yang akan diobservasi dan melakukan validasi pertanyaan bersama dosen pengampu untuk uji kelayakan pertanyaan. Melakukan observasi di Sekolah Dasar yang telah ditentukan untuk mendapatkan data umum. Data umum penelitian ini adalah hasil wawancara dari materi yang telah ditetapkan. Data tersebut dianalisis dan dibahas serta pada akhirnya disimpulkan. Berdasarkan teori Mulyasa (2011: 51), mengemukakan agar tercipta suatu hubungan dan kerja sama yang baik antara masyarakat dan sekolah, maka masyarakat perlu mengetahui dan memiliki gambaran yang jelas tentang keadaan sekolah yang bersangkutan. Gambaran tentang sekolah diberitahukan melalui televisi, dan laporan tahunan. Berdasarkan hasil wawancara di sekolah tersebut mendapatkan dukungan masyarakat dalam melaksanakan mutu pendidikan adalah dengan memberikan informasi nyata serta tidak dibuat-buat maka akan mudah diterima oleh masyarakat. Kalau terkait dengan melaksanakan mutu pendidikan, berawal dari hal-hal yang masyarakat rasakan dari sekolah, jadi dengan adanya kepercayaan masyarakat untuk memasukkan anak mereka ke sekolah tersebut serta upaya yang dilakukan humas sekolah meninformasikan kepada publik khususnya dalam penyelenggaraan humas sekolah. Dengan menggunakan media sosial salah satunya media sosial facebook dimana sekolah dapat c. Dokumentasi Dokumentasi adalah catatan peristiwa yang telah berlalu. Dokumentasi bisa berbentuk tulisan, gambar, dan karya-karya monumental seseorang (Sugiyono, 2015:329). Dokumentasi untuk melengkapi mendapatkan data Sehingga peneliti tidak hanya melakukan observasi dan wawancara saja melainkan mengumpulkan dokumentasi yang dibutuhkan dalam penelitian baik berupa sumber tertulis dan gambar. Berdasarkan teori Mulyasa (2011: 50), mengatakan bahwa masyarakat dan sekolah memiliki hubungan yang sangat erat dalam mencapai tujuan sekolah atau pendidikan secara efektif dan efisien. Berdasarkan hasil wawancara kami di sekolah tersebut, program kerjasama masyarakat dengan sekolah memberikan dampak positif yaitu sekolah lebih terbuka dalam memberikan informasi tentang sekolah dan dari program sekolah yang telah dilakukan, sekolah lebih merasakan respon positifnya dari masyarakat dibandingkan respon negatifnya. Jadi sekolah tersebut sudah merasakan respon positif tentang program kerjasama antara hubungan masyarakat dengan sekolah. b. Wawancara Wawancara yaitu aktivitas percakapan yang dilakukan oleh dua orang dengan maksud tertentu yang terdiri dari orang yang mewawancara yang mengajukan pertanyaan, kemudian dijawab oleh orang yang diwawncarai. HASIL DAN PEMBAHASAN Tarakan. Observasi dilakukan untuk memperoleh data yang sesuai dengan penelitian. Berdasarkan kajian teori, hubungan yang harmonis humas dengan sekolah ini semakin dirasakan pentingnya pada masyarakat yang telah menyadari betapa pendidikan sangat penting. Sedangkan berdasarkan hasil wawancara kami di sekolah ini maka dapat diketahui bahwa cara menjaga hubungan masyarakat dengan sekolah agar tetap terjalin baik yaitu dengan menjaga komunikasi agar tetap berjalan baik antara sekolah dan masyarakat, sebaliknya masyarakat juga membantu sekolah agar tetap terjalin silaturahmi yang baik. Jadi sekolah ini sudah menerapkan cara menjaga hubungan masyarakat dengan sekolah agar tetap terjalin baik. JURNAL BORNEO HUMANIORA Available online at jurnal.borneo.ac.id Diterbitkan Agustus 2019 Halaman 01-07 e-ISSN 2599-3305 http://jurnal.borneo.ac.id/index.php/borneo_humaniora a. Observasi Faisal dalam Sugiyono (2015: 310) observasi digolongkan dalam tiga macam yaitu participant observation, overt observation and covert observation, unstructured observation. Hubungan sekolah dengan masyarakat merupakan segala kegiatan yang berhubungan dengan kegiatan sekolah dengan masyarakat termasuk salah satunya ialah orang tua siswa yang bertujuan untuk membantu dan menunjang kegiatan belajar mengajar disekolah tersebut. Semua perencanaan kegiatan diperlukan adanya humas dengan sekolah untuk memudahkan dalam pemberian informasi kepada masyarakat. Dalam penelitian ini teknik yang digunakan yaitu observasi terus terang atau terbuka. Dalam hal ini, peneliti secara langsung melakukan penelitian kepada sumber data, peneliti sedang melakukan penelitian. Peneliti mengobservasi masing-masing satu sekolah untuk satu lokal, diantaranya Lokal A 2016 di SDIT Ulul Albab Donna Rhamdan, Jumriani, Diana Perminas, Satriani DESKRIPSI HUBUNGAN MASYARAKAT… Donna Rhamdan, Jumriani, Diana Perminas, Satriani DESKRIPSI HUBUNGAN MASYARAKAT… Page 2 Donna Rhamdan, Jumriani, Diana Perminas, Satriani DESKRIPSI HUBUNGAN MASYARAKAT… JURNAL BORNEO HUMANIORA e-ISSN 2599-3305 p-ISSN 2615-4331 http://jurnal.borneo.ac.id/index.php/borneo_humaniora JURNAL BORNEO HUMANIORA e-ISSN 2599-3305 p-ISSN 2615-4331 http://jurnal.borneo.ac.id/index.php/borneo_humaniora dalam hal peraturan yang telah ditentukan oleh sekolah. Bentuk pelaksanaan program kerja sekolah ada yang terlaksana secara rutin, secara tahunan dan berkelanjutan, jenis-jenis kegiatan antara masyarakat dengan sekolah yang telah terlaksana yaitu berupa kegiatan tahunan yaitu pemotongan hewan kurban, kemudian mempunyai program di bulan Ramadhan misalnya siswa membagikan takjil langsung kerumah-rumah tetangga. Selain itu juga sekolah telah melaksanakan kegiatan berupa bantuan misalnya kalau ada musibah contohnya kebakaran,bebarapa siswa melihat langsung dan memberikan bantuan langsung. Jadi bentuk hubungan kerjasama antara sekolah dan masyarakat sudah terjalan dengan baik dan bentuk pelaksanaan program kerja dari humas sekolah telah berkesinambungan. memanfaatkan media tersebut untuk memperkenalkan sekolah tersebut kepada masyarakat serta sekolah mempunyai waka humas dan waka humas tersebut tidak mempunyai kerja sampingan dan hanya fokus di bagian humas saja. Jadi, sekolah sudah melakukan cara yang sesuai untuk mendapatkan dukungan dari masyarakat yaitu dengan memberikan informasi yang nyata, sehingga terciptanya komunikasi yang baik. Dalam menarik simpati masyarakat dengan menggunakan media sosial untuk memperkenalkan sekolah tersebut kepada masyarakat. y Berdasarkan teori Rugaiyah dalam Fadiyah (2016:13) hubungan antara sekolah dengan masyarakat di definisikan sebagai proses komunikasi antara sekolah dan masyarakat yang bertujuan untuk menanamkan pengertian warga masyarakat terkait kebutuhan serta pendorong minat dan tanggung jawab masyarakat dalam upaya memajukan sekolah. Berdasarkan hasil wawancara kami di sekolah tersebut, dukungan masyarakat dalam pelaksanaan peraturan sekolah adalah terlaksananya peraturan sekolah, dengan ikut serta untuk melaksanakan dan ikut andil dalam peraturan yang telah ditetapkan contohnya seperti peraturan dimana siswa di sekolah tersebut dilarang untuk membawa uang saku saat kesekolah dan pihak masyarakat pun mengikuti aturan tersebut. Jadi masyarakat mendukung pelaksanaan peraturan sekolah misalnya siswa dilarang untuk membawa uang saku kesekolah, dan sekolah sudah mendapatkan persetujuan dari warga dan masyarakat sekitar sekolah tentang peraturan tersebut. Visi sekolah tersebut adalah menjadi sekolah yang terbaik dan Islami di Kalimantan Utara. Misi sekolah tersebut adalah 1) mampu menumbuhkan budaya Islami di lingkungan sekolah; 2) mampu mengedepankan qudwah hasanah dalam membentuk karakter peserta didik; 3) mampu pembelajaran yang jauh lebih kreatif; menyenangkan dan berkualitas; 4) mampu menumbuhkan semangat berprestasi pada seluruh warga sekolah; 5) suasana sekolah yang ceria serta kondusif; 6) komunikasi yang menyenangkan dan efektif; 7) mengembangkan bakat dan minat, dikegiatan ekstrakurikuler; 8) mampu membangun budaya sehat, asri, ringkas, runut, resik rapih dan rawat; 9) mengintegrasikan kurikulum, metodologi, dan program yang berkesinambungan; 10) melaksanakan kerjasama dengan stakeholder. e. Analisis Data Penelitian ini mengunakan pendekatan kualitiatif dengan dengan teknik pengumpulan data melalui dokumen, penentuan pola analisis data tergantung jenis data yang dikumpulkan. Dikarenakan jenis penelitian yang dilakukan adalah penelitian kepustakaan, maka data yang diperoleh adalah textular dan pola analisis yang dilakukan adalah analisis non-statistik. Data yang dianalisis menurut isinya, maka teknik analisis yang dapat digunakan adalah teknik content analysis. Menurut Burhan (2008:231) analisis isi adalah teknik penelitian membuat inferensi yang dapat ditiru, dengan memperhatikan konteksnya. Analisis isi berhubungan dengan komunikasi atau isi komunikasi pada penelitian ini peneliti menganalisis humas di SDIT Ulul Albab Tarakan. Donna Rhamdan, Jumriani, Diana Perminas, Satriani DESKRIPSI HUBUNGAN MASYARAKAT… Donna Rhamdan, Jumriani, Diana Perminas, Satriani DESKRIPSI HUBUNGAN MASYARAKAT… Page 3 Available online at jurnal.borneo.ac.id Diterbitkan Agustus 2019 Halaman 01-07 JURNAL BORNEO HUMANIORA e-ISSN 2599-3305 p-ISSN 2615-4331 http://jurnal.borneo.ac.id/index.php/borneo_humaniora JURNAL BORNEO HUMANIORA e-ISSN 2599-3305 p-ISSN 2615-4331 http://jurnal.borneo.ac.id/index.php/borneo_humaniora kegiatan dan program dari SDIT Ulul Albab Tarakan, kemudian juga komunikasi yang dilakukan humas mengenai cara komunikasi sekolah dengan orang tua, misalnya tanggapan- tanggapan serta sosialisasi mengenai program kerja dari sekolah. Jadi pada saat tujuan humas diterapkan terutama pada poin memberikan informasi kepada masyarakat tentang isi dan pelaksanaan program sekolah, akan menilai kinerja humas dengan melihat hal-hal yang dipublikasikan atau di share humas terkait kegiatan dan program dari sekolah. masyarakat yang diperoleh dari sekolah adalah a) sekolah dapat termotivasi untuk terus melakukan perbaikan baik dari segi tenaga pendidik maupun dari fasilitas pedidikan, b) sekolah dapat menyampaikan mengenai kesulitan-kesulitan yang sedang dialami sekolah yang memerlukan partisipasi masyarakat untuk menyelesaikannya, c) sekolah dapat memberikan informasi dan pemahaman kepada masyarakat tentang konsep pendidikan supaya terhindar dari kesalah pahaman antara sekolah dengan masyarakat, d) sekolah dapat menggunakan masyarakat sebagai salah satu sumber belajar bagi siswa. Berdasarkan hasil wawancara kami di sekolah tersebut, manfaat dari kerjasama sekolah dan masyarakat yang dilakukan di sekolah yaitu masyarakat bisa menerima keadaan sekolah, proses kerjasama bisa berjalan dengan baik, kegiatan bisa terlaksanakan. Jadi adapun manfaat dari humas dengan sekolah sudah berjalan, salah satunya bisa menerima keadaan sekolah yaitu pada poin a) sekolah melaksanakan peningkatan kualitas guru. Berdasarkan teori T.Sianipar dalam Purwanto, menyatakan bahwa jika ditinjau dari kebutuhan masyarakat itu sendiri, tujuan adanya hubungan masyarakat dengan sekolah yaitu salah satunya adalah untuk mendapatkan bantuan sekolah dalam memecahkan masalah yang sedang dihadapi masyarakat. Berdasarkan hasil wawancara kami di sekolah tersebut jika masyarakat dan sekolah telah sepakat mengadakan kegiatan maka dana dan fasilitas penunjanganya berasal dari sekolah. Jadi kegiatan hubungan masyarakat bertujuan untuk memperoleh bantuan dana dari sekolah dan di sekolah tersebut telah menerapkan tujuan hubungan masyarakat dengan sekolah dalam problem solving yang dihadapi oleh masyarakat salah satunya dalam melakukan kegiatan menggunakan dana dari sekolah dan dana tersebut digunakan untuk kebutuhan sekolah dan juga melengkapi fasilitas-fasilitas sekolah. p g g Berdasarkan teori Menurut Rahmad (2016:124), dengan adanya humas dengan sekolah berjalan dengan baik akan memberi manfaat pada kedua pihak. Dari hasil wawancara kami di sekolah tersebut, timbal balik dari sekolah yaitu masyarakat berkonstribusi dalam kegiatan yang dilakukan oleh pihak sekolah antara lain program- program di bulan Ramadhan, bersilahturahmi jika ada yang mengundang, lalu misalnya ada acara 17 Agustus jika Sekolah diberikan proposal maka sekolah akan memberikan bantuan dana. JURNAL BORNEO HUMANIORA e-ISSN 2599-3305 p-ISSN 2615-4331 http://jurnal.borneo.ac.id/index.php/borneo_humaniora Pasal 4 peraturan pemerintah (PP) Nomor 39 Tahun 1992 yang meliputi 1) melibatkan wali murid dalam suatu hal yang menunjang pelaksanaan pendidikan, 2) pemberian bantuan tenaga ahli, 3) mengusahakan agar tokoh-tokoh masyarakat dapat turut menunjang pelaksanaan pendidikan, 4) pengadaan dana dan memberi bantuan yang berupa wakaf, beasiswa, hibah, pinjaman dan bentuk-bentuk lain, 5) peyediaan buku pelajaran serta peralatan pendidikan lain untuk melaksanakan kegiatan belajar mengajar. Berdasarkan hasil wawancara kami di sekolah tersebut, bentuk hubungan kerjasama sekolah dan masyarakat sudah terjalin salah satunya yaitu kontribusi masyarakat sekitar dan orang tua murid dalam setiap pelaksanaan kegiatan yang dilakukan sekolah serta bentuk kesepakatan orang tua murid Berdasarkan hasil wawancara kami tujuan utama humas Sekolah tersebut sudah sesuai dengan visi dan misi dari sekolah tetapi masih dalam proses pencapaian. Jadi berdasarkan visi dan misi dengan hasil wawancara, tujuan utama humas dengan sekolah sudah sesuai dengan visi dan misi dari sekolah tersebut meskipun masih dalam proses pencapaian. Berdasarkan teori Rahmad (2016: 124) terjalinnya hubungan antara sekolah dan masyarakat memiliki tujuan salah satunya yaitu memberikan informasi kepada masyarakat tentang isi serta pelaksanaan program sekolah. Berdasarkan hasil wawancara kami di sekolah tersebut, prosedur sekolah tersebut dalam menilai kinerja humas dapat dilihat langsung dari hal-hal yang dipublikasikan atau di share humas terkait Donna Rhamdan, Jumriani, Diana Perminas, Satriani DESKRIPSI HUBUNGAN MASYARAKAT… Donna Rhamdan, Jumriani, Diana Perminas, Satriani DESKRIPSI HUBUNGAN MASYARAKAT… Page 4 Available online at jurnal.borneo.ac.id Diterbitkan Agustus 2019 Halaman 01-07 JURNAL BORNEO HUMANIORA e-ISSN 2599-3305 p-ISSN 2615-4331 http://jurnal.borneo.ac.id/index.php/borneo_humaniora Donna Rhamdan, Jumriani, Diana Perminas, Satriani DESKRIPSI HUBUNGAN MASYARAKAT… JURNAL BORNEO HUMANIORA e-ISSN 2599-3305 p-ISSN 2615-4331 http://jurnal.borneo.ac.id/index.php/borneo_humaniora JURNAL BORNEO HUMANIORA e-ISSN 2599-3305 p-ISSN 2615-4331 http://jurnal.borneo.ac.id/index.php/borneo_humaniora hubungan, yaitu berupaya membina hubungan yang positif serta saling menguntungkan kedua belah pihak; 3) Peranan pendukung pariwisata, yaitu sebagai pendukung dalam fungsi pariwisata organisasi; 4) Membentuk citra coorporasi, artinya peranan humas berupaya untuk menciptakan citra yang baik bagi organisasi. Berdasarkan hasil wawancara kami dengan sekolah tersebut, peran humas sangat penting dalam aktivitas sekolah, dimana peran humas sebagai penyambung informasi disekolah antara kepala sekolah dengan komite-dengan masyarakat. Peran humas di sekolah tersebut dalam meningkatkan kinerja sekolah yaitu membangun sistem kekeluargaan dan kebersamaan misalnya mengadakan rapat santai antara guru dengan wali peserta didik. SDIT Ulul Albab Tarakan telah menerapkan peran humas yang sesuai dengan salah satu peran humas yaitu sebagai komunikator atau penghubung antara organisasi. humas yang sesuai dengan publicity: berkaitan dengan fungsi marketing yang mempublikasikan hal-hal positif mengenai klien atau karyawan menggunakan media sosial. humas yang sesuai dengan publicity: berkaitan dengan fungsi marketing yang mempublikasikan hal-hal positif mengenai klien atau karyawan menggunakan media sosial. gg Berdasarkan teori Seitel dalam Fadiyah (2016: 16-17) fungsi humas yaitu: a) menulis merupakan keahlian dari seorang humas dan merupakan ruang lingkup dari humas, b) seorang humas berkerjasama melakukan publisitas di media massa, c) merencanakan berbagai macam kegiatan, d) menyediakan sarana, e) humas meneliti sikap dan opini yang mempengaruhi perilaku dan kepercayaan masyarakat. Dari hasil wawancara kami disekolah tersebut, guru yang ditunjuk sekolah sebagai humas sekolah memiliki kompetensi di bidang humas sekolah, antara lain dapat berkomunikasi dengan baik, mampu memberikan informasi yang jelas terkait program- program yang ada di sekolah, mempunyai kemampuan bekerja sama, mempunyai kemampuan dalam menggunakan media sosial, memiliki wawasan yang luas, serta berpenampilan yang rapi dan menarik. Jadi fungsi humas adalah memiliki keahlian dalam menulis, mampu bekerja sama, memiliki sikap yang santun dan baik. Humas di sekolah tersebut telah menunjuk waka humas sesuai dengan kemampuan dan keahlian yang dimiliki, dan sudah sesuai dengan fungsi humas dengan sekolah. JURNAL BORNEO HUMANIORA e-ISSN 2599-3305 p-ISSN 2615-4331 http://jurnal.borneo.ac.id/index.php/borneo_humaniora Jadi dari hubungan masyarakat dengan sekolah sudah terjalin dengan baik dan dalam melakukan program-program kerjasama yang ada sudah memberikan manfaat dan timbal balik bagi masyarakat dan juga sekolah tersebut. g p Berdasarkan teori T.Sianipar dalam Purwanto, salah satu tujuan hubungan masyarakat dengan sekolah yaitu untuk memperoleh dukungan serta bantuan dari masyarakat yang diperlukan dalam pengembangan dan pelaksanaan program pendidikan yang dilaksanakan di sekolah. Dari hasil wawancara kami disekolah tersebut, sekolah mengadakan kegiatan selalu mendapat respon positif dari masyarakat, baik masyarakat sekitar maupun orang tua siswa selalu memberikan respon positif, respon itu dapat berupa mendukung acara tersebut selain itu pihak masyarakat juga menyimpan kepercayaan serta kepeduliaan terhadap sekolah ini. Jadi tujuan hubungan masyarakat dan sekolah adalah mendapatkan dukungan dari masyarakat dan sekolah tersebut telah mendapatkan dukungan dari masyarakat berupa respon positif yang baik dan kepercayaan masyarakat kepada sekolah tersebut. Berdasarkan teori Seitel dalam Fadiyah (2016: 16-17) humas memiliki fungsi tersendiri dalam sebuah organisasi, yaitu publicity: berkaitan dengan fungsi marketing yang mempublikasikan hal-hal positif mengenai klien atau karyawan melalui media tertentu dalam keterkaitan yang lebih erat. Berdasarkan hasil wawancara kami di sekolah tersebut penyelenggaraan humas di sekolah yaitu setiap mengadakan kegiatan selalu dipublikasikan oleh humas agar sekolah diketahui oleh masyarakat sekitar terkait dengan kegiatan yang dilakukan baik itu kegiatan rutin yang telah dilakukan setiap hari maupun kegiatan tahun. Jadi, penyelenggaran humas termasuk dalam fungsi Berdasarkan teori Rahmad (2016 :124), adapun manfaat dari hubungan sekolah dengan Donna Rhamdan, Jumriani, Diana Perminas, Satriani DESKRIPSI HUBUNGAN MASYARAKAT… Donna Rhamdan, Jumriani, Diana Perminas, Satriani DESKRIPSI HUBUNGAN MASYARAKAT… Page 5 Available online at jurnal.borneo.ac.id Diterbitkan Agustus 2019 Halaman 01-07 JURNAL BORNEO HUMANIORA JURNAL BORNEO HUMANIORA e-ISSN 2599-3305 p-ISSN 2615-4331 http://jurnal.borneo.ac.id/index.php/borneo_humaniora komunikasi tatap muka. Berdasarkan hasil wawancara, media yang digunakan yaitu melalui media sosial. Jadi, media yang digunakan dengan sekolah sudah sesuai dengan 2 media yang ada, yaitu media siaran (broadcast media). Fadiyah, Rozanah Ahlam. 2016. Peran Humas Dalam Membangun Citra Positif Sekolah Di Sd N Sosrowijayan Yogyakarta. Yogyakarta: Universitas Negeri Yogyakarta. Skripsi (Online) Imaniyah, Rizky dkk.2016. Pengelolaan Hubungan Sekolah Dan Masyarakat Home- Schooling. Jurnal Manajemen dan Supervisi Pendidikan. Vol. 1 No. 1 JURNAL BORNEO HUMANIORA e-ISSN 2599-3305 p-ISSN 2615-4331 http://jurnal.borneo.ac.id/index.php/borneo_humaniora g Menurut pendapat Indra fachrudi dalam Yulitasari (tanpa tahun: 13-14) tentang beberapa teknik yang telah dilaksanakan di Indonesia, yaitu : a) Group meeting (pertemuan kelompok) yang meliputi temu fakta, diskusi, bekerja sambil bermain bersifat rekreasi yang berbentuk pertemuan keluarga; b) Face to face (pertemuan tatap 14 muka) yang meliputi kunjungan ke rumah peserta didik dan penyampaian kepada wali peserta didik; c) Observation and participation (observasi dan partisipasi) yang meliputi orang tua sebagai observer, orang tua sebagai peserta dan ibu pembantu kelas; d) The written word (berucap di kertas) yang meliputi catatan berita gembira, berita dalam surat, buku kecil permulaan sekolah, dan pamphlet. Berdasarkan hasil wawancara kami di sekolah, teknik yang digunakan berawal dari program kerja sekolah misalnya program kerja sekolah yang sifatnya kemasyarakatan, jadi ada arah untuk humas sekolah mengaturnya contoh program yang sifatnya setahun sekali. Misalnya sekolah mengadakan rapat pertemuan membahas kegiatan yang akan dilaksanakan dengan wali peserta didik. Jadi, dari beberapa teknik yang ada bahwa sekolah ini sudah menerapkan salah satu teknik yaitu Group meeting (pertemuan kelompok). Sekolah ini mengadakan rapat membahas kegiatan yang akan dilaksanakan. g Berdasarkan teori Qoimah (2018: 199) fungsi humas yaitu mengikut sertakan masyarakat dalam usaha mengatasi berbagai persoalan pendidikan serta meningkatkan kerjasama humas dengan sekolah. Berdasarkan Hasil observasi kami disekolah tersebut, perbedaan pendapat antara masyarakat dan juga sekolah dalam hal mencapai tujuan bersama berjalan dengan baik, sekolah membuka diri apabila ada masukan dari masyarakat dan apabila kalau ada sampai komplain atau keberatan dari masyarakat itu tidak ada. Jadi fungsi humas adalah untuk mengatasi persoalan hubungan masyarakat dengan sekolah untuk mencapai tujuan bersama dan di sekolah sudah berjalan denganbaik fungsi humas sehingga masyarakat tidak keberatan dan tidak ada yang kompalin serta masyarakat menerima dengan baik. Berdasarkan teori Rosady Ruslan dalam Cahyaningsih (2015:13) menjelaskan secara rinci empat peran utama humas adalah sebagai berikut : 1) Sebagai komunikator antara organisasi dan lembaga yang diwakilinya; 2) Membina Menurut F. Rachmadi Cahyaningsih (2015:17) menjelaskan tentang media komunikasi yang digunakan oleh organisasi humas meliputi : 1) Media berita, 2) Media siaran 3) Media Donna Rhamdan, Jumriani, Diana Perminas, Satriani DESKRIPSI HUBUNGAN MASYARAKAT… Page 6 Available online at jurnal.borneo.ac.id Diterbitkan Agustus 2019 Halaman 01-07 JURNAL BORNEO HUMANIORA e-ISSN 2599-3305 p-ISSN 2615-4331 http://jurnal.borneo.ac.id/index.php/borneo_humaniora JURNAL BORNEO HUMANIORA e-ISSN 2599-3305 p-ISSN 2615-4331 http://jurnal.borneo.ac.id/index.php/borneo_humaniora KESIMPULAN Sekolah ini telah melakukan cara yang sesuai untuk mendapatkan dukungan dan simpati dari masyarakat, yaitu dengan memberikan informasi yang nyata melalui media social, sehingga peran humas dengan sekolah yaitu menjaga hubungan baik. Manfaat dari bentuk kerjasama humas dengan sekolah sudah berjalan dengan baik serta bentuk atau teknik dalam pelaksanaan program kerja dari humas berkesinambungan. Mulyasa, E. 2011. Manajemen Berbasis Sekolah. Bandung: PT Remaja Rosdakarya Purwanto, Ngalim. 2012. Administrasi dan Supervisi Pendidikan. Bandung: PT Remaja Rosdakarya Qoimah. 2018. Strategi Manajemen Humas Dalam Penyampaian Program Unggulan Di Lembaga Pendidikan. Jurnal Manajemen Pendidikan. Vol. 1 No. 2 Tujuan hubungan masyarakat dan sekolah adalah mendapatkan dukungan dari masyarakat dan sekolah tersebut telah mendapatkan dukungan dari masyarakat berupa respon positif yang baik dan kepercayaan dari masyarakat. Bentuk penyelenggaraan humas merupakan fungsi dari humas dengan sekolah yaitu untuk mengatasi persoalan hubungan masyarakat dengan sekolah sehingga tercapai tujuan bersama dan di sekolah ini sudah berjalan dengan baik fungsi humas sehingga masyarakat tidak keberatan dan tidak ada yang complain serta masyarakat menerima dengan baik. Rahmad, Abdul. 2016. Manajemen Humas Sekolah. Yogyakarta: Media Akademi Ratri, Safitri. 2009. Kerjasama Sekolah Dengan Masyarakat Dalam Manajemen Peningkatan Mutu Di Sekolah Dasar Se Kecamatan Pakualaman Yogyakarta. Yogyakarta. Universitas Negeri Yogyakarta. Penelitian (Online) Sugiyono. 2015. Metode Penelitian Pendidikan (Pendekatan Kuantitatif, Kualitatif, dan R&D). Bandung: Alfabeta g Yulitasari, Effa. 2015. Pelaksanaan Hubungan Sekolah dan Masyarakat di Sekolah Berbasis Pesantren (Studi Kasus Di SMP Ar-Rohmah Putri Malang). Malang: Universitas Negeri Malang. Skripsi (Online) DAFTAR PUSTAKA Bungin, Burhan. 2008. Penelitian kualitatif. Jakarta: Kencana Prenada Media Group Cahyaningsih, Estih. 2015. Peran Humas Dalam Rangka Membangun Citra Dan Mempromosikan Smk Pgri 1 Sentolo Kulon Progo. Yogyakarta: Universitas Negeri Yogyakarta. Skripsi (Online) DAFTAR PUSTAKA DAFTAR PUSTAKA Bungin, Burhan. 2008. Penelitian kualitatif. Jakarta: Kencana Prenada Media Group Cahyaningsih, Estih. 2015. Peran Humas Dalam Rangka Membangun Citra Dan Mempromosikan Smk Pgri 1 Sentolo Kulon Progo. Yogyakarta: Universitas Negeri Yogyakarta. Skripsi (Online) g g p ( ) PP Nomor 39 Tahun 1992 UU No. 20 Tahun 2003 tentang Sistem Pendidikan Nasional Donna Rhamdan, Jumriani, Diana Perminas, Satriani DESKRIPSI HUBUNGAN MASYARAKAT… Donna Rhamdan, Jumriani, Diana Perminas, Satriani DESKRIPSI HUBUNGAN MASYARAKAT… Page 7 Page 7
W2413708648.txt	https://www.mdpi.com/2543-6031/80/4/292/pdf?version=1658816601	en		Opieka nad chorymi na POChP w Danii	Advances in Respiratory Medicine	2,012	cc-by	2,529	ARTYKUŁ PRACA REDAKCYJNY ORYGINALNA Peter Lange Department of Social Medicine, Institute of Public Health, University of Copenhagen Respiratory Section, Hvidovre Hospital Copenhagen City Heart Study, Bispebjerg Hospital Chronic care for COPD patients in Denmark Opieka nad chorymi na POChP w Danii No financial support received. Pneumonol. Alergol. Pol. 2012; 80, 4: 292–295 COPD in Denmark The prevalence of chronic obstructive pulmonary disease (COPD) in Denmark is among the highest in the world [1]. This is mainly caused by a very high prevalence of smoking in both men and women throughout the latest 5 decades. Although the percentage of daily smokers among adult Danes has steadily declined to around 20% in 2011, COPD and other smoking related conditions are still a major health problem in Denmark. Based on spirometric findings from the Copenhagen City Heart Study and Copenhagen General Population Study, it has been estimated that around 400,000 Danes have COPD — corresponding to 7% of the total Danish Population, which comprises around 5.4 million [2]. It has also been estimated that around 40,000 individuals have severe or very severe COPD, defined as FEV1 below 50% of the predicted value. The mortality from COPD is also very high in Denmark. In 2010, app. 3,800 deaths were attributed to COPD, and in an additional 2,200 deaths COPD was acknowledged as a contributory factor, resulting in a total of app. 5,000 deaths, corresponding to app. 10% of all deaths [3]. The number of visits in general practice due to COPD is unknown, since Danish general practitioners, until now, have not performed any routine diagnostic coding describing the type of contact (diagnosis of the patient) in their practice. The drain of COPD on the Danish hospitals is, however, well described by means of a nationwide register cove- ring all hospital contacts in Denmark. In 2010, there where around 24,000 acute hospital admissions and 53,000 scheduled visits to hospital outpatient clinics due to COPD [4]. In order to improve the care for COPD, the Danish Board of Health and the Danish Regions have launched a disease management program describing optimal care for COPD. These programs describe the role of three major players in Danish society responsible for the delivery of healthcare for individuals with COPD: general practice (family doctors = GPs), hospitals (specialized care), and municipalities [5, 6]. In this short review I will describe the collaboration between these three, with special focus on the role of GPs, but first I will give some brief details on the Danish healthcare system as such. Danish Healthcare System and COPD care All people registered as residents in Denmark are entitled to free healthcare [7, 8]. The healthcare system is almost 100% publicly financed through taxes. This means that consultations with a GP or specialists and all hospital treatment is free of charge, whereas patients have to pay for the drugs that are prescribed to them. With regards to the latter, there are rules for reimbursement of the drug expenditure, according to the type of drug and when the payment attains a certain level. The Danish health system is organised around app. 3,600 GPs who, although being technically private, are financed by public taxes and operate Adres do korespondencji: Prof. Peter Lange, Section of Social Medicine, Øster Farimagsgade 5, P.O.B. 2099, DK-1014 Copenhagen K. e-mail: Peter.lange@sund.ku.dk Praca wpłynęła do Redakcji: 12.01.2012 r. Copyright © 2012 Via Medica ISSN 0867–7077 292 www.pneumonologia.viamedica.pl Peter Lange, Chronic care for COPD patients in Denmark entirely within the public healthcare system. They act as gatekeepers to specialists and to the hospitals. This, in practice, means that in order to consult a pulmonologist, either in private practice or at a hospital, patients have to be referred by their own GP [7, 8]. The idea is that the GPs should provide most of the care since they are cheaper. The number of pulmonologists serving the whole Danish population is around 120, and more than 90% of them are hospital-based, working at app. 70 public hospitals organised under the five Danish Regions, each covering from 0.5 to app. 1.5 million inhabitants. The waiting time from a referral from the GP to a pulmonologist varies between the different regions of Denmark, but there is a fast track if the chest X-ray gives a suspicion of a malignant disease. Otherwise, the waiting time to see a pulmonologist for COPD is between 2 and 6 months, on average. In addition to the GPs and pulmonologists, the third group of players with regard to COPD are the 98 Danish municipalities. In addition to their main task, which is to run nursing homes, home nurses, health visitors, and school health services, the municipalities share, together with the hospitals, the responsibility for providing the rehabilitation of patients with chronic diseases, including COPD. This means that relevant patients are entitled to a COPD rehabilitation program either at a local training centre in their municipality or at the nearest hospital. The choice of location for rehabilitation depends on the severity of COPD: moderate and severe COPD is often rehabilitated in a municipality-based health centre, whereas in very severe COPD the rehabilitation program is performed at the hospital. The acknowledgement that chronic diseases will continue to be a major health problem for many decades to come led, in 2007, to the introduction of “the national strategy for chronic disease management”, by the Danish Board of Health [9]. This system, which is mainly inspired by the Chronic Care Model and organisations like Kaiser Permanente [10], is based on several pillars including, among others: early identification of individuals with chronic disease, self-management support, stratification according to disease severity in order to define where the patient should be treated and followed-up, distribution of tasks between the GP, hospital-based specialist, and the municipality, and quality assurance. The description of optimal care for COPD according to this concept has been done on regional level during the last three years and has been supported by binding agreements between the hospitals, general practice, and the local municipalities. Quality assurance systems are currently being launched in order to evaluate the effect of these programs and to monitor the quality of care delivered to the individual COPD patients Quality of COPD care in Denmark: past and future Some information on the quality of COPD care in Denmark is available. In general practice a few studies have been performed focusing, among others, on the use of spirometry, correct medication, assessment of dyspnoea by means of the MRCdyspnoea scale, and assessment of body mass index (BMI) [11, 12]. These studies, based on relatively large samples of GPs, have shown that quality was not optimal, e.g. only about 50% of patients treated with bronchodilators for COPD in general practice had spirometry performed. However, these studies have also shown that the quality of care can be improved by providing education programs to the GPs. A similar study of the quality of care delivered to COPD patients treated as out-patients in Danish hospitals showed that although the frequency of spirometry was satisfactory, but the focus on objective measurements of dyspnoea (the use of the MRC-dyspnoea scale), BMI, and on the comorbidities was too low [13]. Consequently, many initiatives to monitor and improve the quality of COPD care in both general practice and in the hospitals have been initiated throughout Denmark. Since 2008, all Danish hospitals have to monitor the quality of their COPD care. This means that they are continuously obliged to report data into a central COPD database. The data comprise several indicators of good COPD care, including: spirometry, BMI, smoking status, smoking cessation treatment, and COPD rehabilitation. Since 2008 an annual report covering all data has been produced allowing for observation of a time trend and for benchmarking between different regions and different hospitals [14]. Although the database is not complete at present, it covers more than 70% of all hospital contacts due to COPD, thereby comprising more than 20,000 patients with COPD and more than 30,000 hospital contacts each year. The results have shown a very satisfactory improvement with regard to fulfilment of most of the quality indicators all over Denmark [14]. The annual report is published by a national steering committee and is made available to both participating hospitals and politicians responsible for the regional healthcare system in order to further improve the quality of care. A popularized version of the report has also been made available to the general public [15]. www.pneumonologia.viamedica.pl 293 Pneumonologia i Alergologia Polska 2012, tom 80, nr 4, strony 292–295 Table 1. Stratification of COPD patients Table 1. Stratyfikacja chorych na POChP Criterion Recommended stratification Level 1 General practice FEV1 (in % of predicted when stable) Level 2 Alternating Level 3 Hospital out-patient clinic Mild and moderate COPD Severe COPD Very severe COPD FEV1 ≥ 50% of predicted 30% < FEV1 < 50% of predicted FEV1 ≥ 30% of predicted 1–2 3 4–5 MRC (dyspnoea) All abbreviations in the text/Objaśnienia skrótów w tekście Beginning in 2012, all Danish GPs will also have to report to this COPD database. A set of quality indicators regarding treatment of COPD in general practice has been developed. These indicators are very similar to those used in the hospitals and are based on Danish and international COPD-guidelines. In practice, in connection with the scheduled once-a-year visit focusing on the status of COPD, the GP has to fill in an electronic questionnaire with elements describing the status of the patient including spirometry, assessment of dyspnoea (MRC-dyspnoea scale), smoking status, BMI, inhalation technique, influenza vaccination status, the frequency of exacerbations, and the need for pulmonary rehabilitation. The information is stored on the GPs computer but is also collected centrally to the COPD database through a so-called data-catch module (a search engine designed to collect and interpret the COPD data in the GPs electronic system). Subsequently, the GP can obtain reports describing all his/her COPD patients and the fulfilment of the COPD indicators. This data collection is possible because all Danish GPs have electronic medical software systems on their computers to register patient-related activities, refer the patients to hospitals and specialists, and to send electronic bills to regions in order to receive payment for their work. The future will show how fast the reporting of COPD indicators is implemented among Danish GPs. Cooperation between GPs and pulmonologists The major breakthrough with regard to COPD treatment in general practice in Denmark is the fact that from this year on, all Danish GPs are committed to register their contacts by using diagnosis of the patients according to the international coding for general practice [16]. This allows for the collection of quality data not only for COPD, but also for diabetes and other chronic conditions. The division of tasks between GPs and specialists regarding COPD patients has only recently 294 been defined in the management program for COPD and is shown in the table 1. In general, individuals with GOLD 1–2 disease should be treated by GPs, those with GOLD 4 should be followed by specialists, and those with GOLD 3 constitute a grey area and can be followed by either, depending on the severity of symptoms and frequency of exacerbations. Especially in this group, the concept of shared care between the GP and the specialist is relevant. This, however, depends on good and fast possibilities of communication between the two. At present hospitals can electronically inform the GP on the condition of the patient right after the hospital consultation, whereas it cannot be done the other way round. It is, however, hoped that fast and secure electronic communication pathways between GPs and the hospitals will be developed shortly. In general, it is presently unknown whether the stratification described in the Table is working satisfactorily. Most importantly, it assumes that COPD is well defined and diagnosed with certainty, and this can sometimes be problematic. Future audits will show whether or not Table 1 represents a good division of work between the GPs and the pulmonologists. The stratification also assumes that early diagnosis of COPD in general practice is actually taking place. This is at present uncertain, and a Danish study in general practice has revealed that up to 40% of patients with spirometrydefined severe and very severe COPD are not on inhaled medications, suggesting substantial deficiencies in the diagnostic process [17]. Since Danish GPs have quite good access to spirometry, it is hoped that spirometry will be more widely employed in the future, and new studies indicate that using the criteria of the Danish Board of Health for early diagnosis of COPD is worthwhile [18]. In conclusion, in recent years there has been a substantial focus on the improvement of COPD care in Denmark. Disease management programs defining optimal care and describing the division www.pneumonologia.viamedica.pl Peter Lange, Chronic care for COPD patients in Denmark of tasks between the GPs, the specialists, and the municipalities have been launched and are at present being implemented throughout Denmark. Powerful monitoring tools have been established to follow the quality of care for COPD patients both in general practice and in hospitals. The future will show whether or not these programs and efforts will have a positive impact on morbidity and mortality in the long term. Conflict of interest 6. 7. 8. 9. 10. 11. 12. Author does not declare current or perceived conflict of interest. 13. References 14. 1. 2. 3. 4. 5. Fabricius P., Løkke A., Marott J., Vestbo J., Lange P. Prevalence of COPD in Copenhagen. Respir. Med. 2011; 105: 410–417. Lange P., Marott J.L., Dahl M., Ingebrigtsen T.S., Vestbo J., Nordestgaard B.G. Substantial need for early diagnosis, rehabilitation and treatment of COPD. Danish Medical Journal (e-pub). Death causes in Denmark. http://www.sst.dk/Indberetning% 20og%20statistik/Sundhedsdata/Doedsaarsager/DSN The Danish National Patient Registry. http://www.sst.dk/ Indberetning%20og%20statistik/Landspatientregisteret (14 September 2011) Sundhedstyrelsen. KOL — anbefalinger for tidlig opsporing, opfølgning, behandling og rehabilitering. http://www.sst.dk/ publ/Publ2007/CFF/KOL/KOLanbefalinger.pdf 15. 16. 17. 18. Region Hovedstadens Forløbsprogram for KOL. http://www.regionh. dk/NR/rdonlyres/5B1B6F6B-B369-4636-82DF-01785D198CCE/0/ /Forloebsprogram_KOL_LR.pdf 1 December 1. 2011 Background briefing. Health care lessons from Denmark. http:// www.civitas.org.uk/pdf/Denmark.pdf Vrangbaek K. The Danish Health Care System. http:// www.commonwealthfund.org/~/media/Files/Resources/2008/ Health%20Care%20System%20Profiles/Denmark_Country_ _Profile_2008%20pdf.pdf Danish Board of Health. http://chronisante.inist.fr/sites/chronique/IMG/pdf/chronic_disease_management_Danemark.pdf http://www.improvingchroniccare.org Lange P., Rasmussen F.V., Borgeskov H., Dollerup J., Jensen M.S., Roslind K., Nielsen L.M.; KVASIMODO Study Group. The quality of COPD care in general practice in Denmark: the KVASIMODO study. Prim. Care Respir. J. 2007; 16: 174––178. Ulrik C.S., Hansen E.F., Jensen M.S. et al.; KVASIMODO II study group. Management of COPD in general practice in Denmark—participating in an educational program substantially improves adherence to guidelines. Int J Chron Obstruct Pulmon Dis. 2010; 5: 73–79. Lange P., Andersen K.K., Munch E. et al. Quality of COPD care in hospital outpatients clinics in Denmark: The Kolibri study. Respir. Med. 2009; 103: 1657–1662. National Report regarding the quality of care for COPD in Denmark https://www.sundhed.dk Fil.ashx?id=15199&ext= =pdf&navn=KOL2011_ sfa_national.pdf Quality of care for COPD in Denmark (popular version). https:// www.sundhed.dk/Fil.ashx?id=15198&ext=pdf&navn= =KOL2011_borger_national.pdf Diagnostic coding for general practice. http://www.gpscbc.ca/ system/files/GPSC%20Diagnostic%20Codes.pdf Hansen J.G., Pedersen L., Overvad K., Omland Ø., Jensen H.K., Sørensen H.T. The Prevalence of chronic obstructive pulmonary disease among Danes aged 45–84 years: population-based study. COPD 2008; 5: 347–52. Ulrik C.S., Løkke A., Dahl R. et al.; TOP study group. Early diagnosis of COPD in general practice. Int. J. Chron. Obstruct. Pulmon. Dis. 2011; 6: 123–127. www.pneumonologia.viamedica.pl 295
https://openalex.org/W2004011688	https://www.e-publicacoes.uerj.br/index.php/demetra/article/download/11559/9697	Portuguese	null	DEMANDAS JUDICIAIS PARA FORNECIMENTO DE FÓRMULAS NUTRICIONAIS: LEITURAS DO DIREITO À SAÚDE	Demetra	2,014	cc-by	1,527	Demandas judiciais para fornecimento de fórmulas nutricionais: leituras do direito à saúde Legal claims for the provision of nutritional formulas: readings on the right to health Demandas judiciais para fornecimento de fórmulas nutricionais: leituras do direito à saúde Laura Filomena Santos de Araújo1 Roseney Bellato1 Agradecemos, inicialmente, o convite para nos inserir no debate acerca de demandas judiciais em saúde, em particular no que se refere ao fornecimento de fórmulas nutricionais. Os dados apresentados no artigo “Perfil das demandas judiciais para fornecimento de fórmulas nutricionais encaminhadas ao Ministério da Saúde do Brasil” se mostram bastante abrangentes, podendo gerar inúmeras análises. Optamos, neste debate, por priorizar uma linha argumentativa com base na demanda majoritária de 38,3%, que engloba crianças menores de dois anos de idade, acrescida de 12,1% para crianças entre dois e dez anos de idade, totalizando 50,4% apenas para esse segmento da população. 1 Faculdade de Enfermagem, Universidade Federal de Mato Grosso. Grupo de Pesquisa Enfermagem Saúde e Cidadania. Cuiabá, MT, Brasil. Correspondencia / Correspondence Laura Filomena Santos de Araújo E-mail: laurafil1@yahoo.com.br Essa eleição se justifica, também, por entendermos que nos possibilita aprofundar a perspectiva do direito reiterado da criança à vida e à saúde, tanto garantido na própria Constituição Federativa do Brasil, quanto no Estatuto da Criança e do Adolescente, reverberado em políticas públicas de proteção à criança. Tais políticas, em grande medida propostas e/ou assumidas pelo próprio Ministério da Saúde, como especificado no texto da “Agenda de Compromissos para a Saúde Integral da Criança e Redução da Mortalidade Infantil”,1 visam reforçar o pacto de, em conjunto com estados e Demetra; 2014; 9(Supl.1); 223-226 223 Demetra: alimentação, nutrição & saúde municípios, construir uma rede de assistência pública integral, qualificada e humanizada em benefício da criança brasileira. Vimos na expressão dos dados apontados pelos autores do artigo em debate a possibilidade de trazer para a cena da discussão a responsabilidade do Ministério da Saúde como promulgador de políticas e, essencialmente, ordenador de práticas no campo da saúde, em que pese ser o receptor das demandas emanadas, em grande medida, por Defensorias Públicas (65,6%). Como consequência, torna-se possível também colocar em debate a eficácia dos serviços de saúde na provisão de cuidados integrais à população infantil. Decerto as famílias demandaram, via Defensoria Pública, a garantia daquilo que consideraram necessário para a recuperação da saúde de suas crianças. Demetra; 2014; 9(Supl.1); 223-226 Laura Filomena Santos de Araújo1 Roseney Bellato1 Caberia ao Ministério da Saúde, então, a análise das vulnerabilidades das mesmas, particularmente aquelas menores de dois anos, tanto no sentido do risco, como da ocorrência de desnutrição, dada a significativa vulnerabilidade e altas taxas de mortalidade que ainda recaem sobre essa população, ao que se acresce a condição frágil de saúde decorrente da cronicidade de agravos.2 Tal situação se mostra preponderante no estudo em tela, tal como alergias e intolerâncias alimentares (80,7%), referidas ao grupo das doenças endócrinas, metabólicas e nutricionais. Estudos que realizamos2,3 ressaltam a acolhida das demandas do direito à saúde por parte da magistratura, pesando para isso a vulnerabilidade da pessoa humana nas fases da vida, particularmente quando se trata de crianças em tenra idade. Ademais, a pouca efetividade dos serviços de saúde no acompanhamento das crianças é denotada, no artigo em debate, pela não comprovação diagnóstica (40,5%) das doenças/agravos dos solicitantes, que geram a necessidade das fórmulas nutricionais, particularmente no grupo relacionado às alergias e intolerâncias alimentares (87,0%); e também por não apresentarem a avaliação do estado nutricional do demandante, incluindo o acompanhamento multiprofissional. Tal situação, potencialmente complexa, expõe o componente programático na gênese das vulnerabilidades, sendo esse componente referido ao acesso a serviços, existência e sustentação de programas, qualidade da atenção,4 dentre outros, o qual evidencia a omissão nos deveres do Estado em minimizar danos e riscos à saúde das pessoas. Cabe, portanto, questionar se, de fato, a demanda judicial por fornecimento de fórmulas nutricionais, particularmente para crianças menores de dois anos, tem o condão de comprometer a alocação racional de recursos públicos com agravo das iniquidades de acesso à saúde; ou se demonstra um descompromisso do Ministério da Saúde com essa população, obrigando as famílias a lançar mão dessa via para o cumprimento da garantia do direito à saúde de seus filhos. Demetra; 2014; 9(Supl.1); 223-226 224 Demandas judiciais para fornecimento de fórmulas nutricionais A Defensoria Pública, como instituição provocadora das instâncias do Poder Judiciário, tem por foro privilegiado a defesa dos direitos de parcelas da população consideradas “hipossuficientes”, dentre elas idosos, crianças e adolescentes. Assim, sua intervenção deveria ser compreendida como alerta para o Ministério da Saúde como promulgador de políticas e organizador de práticas que visam a promoção, proteção e recuperação da saúde dessa população. Demetra: alimentação, nutrição & saúde Demetra: alimentação, nutrição & saúde Por fim, ressaltamos que a discussão aqui fomentada está implicada em estudos que temos realizado e que têm por base a defesa do direito à saúde das pessoas, particularmente, como no caso em tela, das crianças. Entendemos que a atenção integral é parte desse direito, sendo a judicialização uma das expressões de sua falta. Assim, frente ao que aqui buscamos dar relevo, é nossa compreensão a premente necessidade de que haja sinergia de esforços3 por parte do Ministério da Saúde e da Defensoria Pública, bem como de outras instâncias defensoras do direito à saúde, no sentido de haver, de fato, efetivação desse direito. Laura Filomena Santos de Araújo1 Roseney Bellato1 Damos destaque, também, aos dados oferecidos pelo artigo ora em tela, que mostram vertiginoso aumento nas demandas judiciais realizadas ao Ministério da Saúde no período de 2007 a 2013, saltando de 39 para 168, respectivamente. Esse interstício de sete anos coincide com o chamado fenômeno da “judicialização da saúde” como espaço público para resolução de conflitos, no qual a provocação e a atuação do Poder Judiciário, ou instâncias provocadoras deste, como a Defensoria Pública e o Ministério Público, agem em prol da efetivação do direito à saúde, através da consecução da assistência em saúde.5 Entendemos que na avaliação pelo setor técnico do Ministério da Saúde, das demandas recebidas por essa instância, deveria ser privilegiada a análise quanto ao acompanhamento em saúde das crianças nos primeiros anos de vida, particularmente aquelas que se encontram sob vulnerabilidade, como é o caso das que são referidas no artigo em debate. Tal análise teria por finalidade o reordenamento de práticas profissionais que pudessem efetivar o que está preconizado nas políticas públicas de atenção à criança e ao adolescente, reiterada, inclusive, por aquelas de cunho mais específico, como é o caso da Política Nacional de Alimentação e Nutrição.6 Faz-se necessário, ainda, questionar que a ocorrência de reiteradas demandas individuais de naturezas semelhantes não gerem, por parte do Ministério da Saúde, a preocupação de tomá-las como demanda coletiva – explicitamente tomamos aqui o fato de, em 2013, ter havido 57 demandas individuais cujos demandantes são crianças menores de dois anos necessitando do fornecimento de fórmula alimentar. Nesse sentido, a judicialização, em sua gênese, pode ter tanto um efeito em cascata fomentador de sua manutenção, reiterando a necessidade de busca pelo “remédio jurídico”7 pela população, quanto de mudanças que consubstanciem a efetividade das práticas profissionais em saúde, através do reordenamento das mesmas no sentido de serem mais integrais e resolutivas. Concernente a tais mudanças, indica-se ao Ministério da Saúde ampliar a análise das demandas judiciais, abarcando elementos analisadores de suas linhas de cuidado integral, no intuito de qualificar o acompanhamento de saúde e, na discussão em voga, garantir a nutrição para a vida saudável. Demetra; 2014; 9(Supl.1); 223-226 225 Recebido: 16/6/2014 Aprovado: 26/6/2014 Referências 1. Brasil. Ministério da Saúde. Agenda de compromissos para a saúde integral da criança e redução da mortalidade infantil. Brasília: Ministério da Saúde; 2004. 2. Mariotto M, Araujo LFS, Bellato R, Dolina JV. Direito à saúde nos discursos do poder judiciário e do poder executivo: análise de decisão judicial. Cogitare Enferm. 2013; 18(4):647-654. 3. Nepomuceno MAS, Bellato R, Araujo LFS, Mufato LF. O campo jurídico na garantia do direito à saúde. R. Dir. Sanit. 2013; 14(2):119-136. 4. Ayres JRCM, França Júnior I, Calazans GJ, Saletti Filho HC. O conceito de vulnerabilidade e as práticas em saúde: novas perspectivas e desafios. In: Czeresnia D, Freitas CM. Promoção da saúde: conceitos, reflexões, tendências. Rio de Janeiro: FIOCRUZ; 2003. p.117-139. 5. Gandini JAD, Barione SF, Souza JE. A judicialização do direito à saúde: a obtenção de atendimento médico, medicamentos e insumos terapêuticos por via judicial: critérios e experiências. Âmbito Jurídico [Internet]. 2008; XI(49). Disponível em: http://www.ambito-juridico.com.br/site/index. php?artigo_id=4182&n_link=revista_artigos_leitura 6. Brasil. Ministério da Saúde. Política nacional de alimentação e nutrição. Brasília: Ministério da Saúde; 2012. 6. Brasil. Ministério da Saúde. Política nacional de alimentação e nutrição. Brasília: Ministério da Saúde; 2012. 7. Bellato R, Araujo LFS, Nepomuceno MAS, Mufato LF. Remédio Jurídico e seus afetamentos no cuidado à saúde de uma família. In: Pinheiro R, Martins PH, organizadores. Usuários, redes sociais, mediações e integralidade em saúde. Rio de Janeiro: UERJ/IMS/LAPPIS; 2011. p.193-201. 7. Bellato R, Araujo LFS, Nepomuceno MAS, Mufato LF. Remédio Jurídico e seus afetamentos no cuidado à saúde de uma família. In: Pinheiro R, Martins PH, organizadores. Usuários, redes sociais, mediações e integralidade em saúde. Rio de Janeiro: UERJ/IMS/LAPPIS; 2011. p.193-201. Recebido: 16/6/2014 Aprovado: 26/6/2014 Demetra; 2014; 9(Supl.1); 223-226 226 226
https://openalex.org/W4247537254	https://www.qeios.com/read/BTHST8/pdf	English	null	Dominica	Definitions	2,020	cc-by	56	Dominica National Cancer Institute National Cancer Institute Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Qeios ID: BTHST8 · https://doi.org/10.32388/BTHST8 Source National Cancer Institute. Dominica. NCI Thesaurus. Code C16519. An island between the Caribbean Sea and the North Atlantic Ocean, south of Guadeloupe and north of Martinique. Qeios ID: BTHST8 · https://doi.org/10.32388/BTHST8 1/1
https://openalex.org/W1836701721	https://figshare.com/articles/journal_contribution/NON-MEMORY-BASED_AND_REAL-TIME_ZEROTREE_BUILDING_FOR_WAVELET_ZEROTREE_CODING_SYSTEMS/13641368/1/files/26189255.pdf	English	null	Non-Memory-Based and Real-Time Zerotree Building for Wavelet Zerotree Coding Systems	Lecture notes in computer science	2,000	cc-by	5,518	1 INTR ODUCTION The w a v elet zerotree co ding systems, including Em b edded Zerotree W a v elet (EZW)[1] and its v arian ts Set P artitioning In Hierarc hical T rees (SPIHT)[2] and Space F requency Quan tization (SF Q)[3][4], ha v e three common pro cedures: 1) 2-D Discrete W a v elet T ransform (D WT)[5], 2) zerotree building and sym b ol generation from the w a v elet co eÆcien ts (illustrated in Figure 1), and 3) quan ti- zation of the magnitudes of signican t co eÆcien ts and en trop y co ding, where the second pro cedure is an imp ortan t one. All recen tly propsed arc hitectures ([6]-[9]) for w a vlelet zerotree co ding use memories to build zerotrees. In this pap er w e con tribute to building the zerotrees in a non-memory-based w a y with real-time p erformance leading to the decrease of hardw are cost and the increasemen t of pro cessing rate whic h is esp ecially desirable in video co ding. One of our main ideas is to rearrange the D WT calculations taking adv an tage of parallel and pip elined pro cessing so that any paren t co eÆcien t and its c hildren co eÆcien ts in zerotrees are guaran teed to b e calculated and outputted sim ultaneously . NON-MEMOR Y-BASED AND REAL-TIME ZER OTREE BUILDING F OR W A VELET ZER OTREE CODING SYSTEMS Dongming P eng and Mi Lu Electrical Engineering Departmen t, T exas A&M Univ ersit y , College Station, TX77843, USA Electrical Engineering Departmen t, T exas A&M Univ ersit y , College Station, TX77843, USA Electrical Engineering Departmen t, T exas A&M Univ ersit y , College Station, TX77843, USA J. Rolim et al. (Eds.): IPDPS 2000 Workshops, LNCS 1800, pp. 469-475, 2000.  Springer-Verlag Berlin Heidelberg 2000 2 THE AR CHITECTURE F OR REARRANGING 2-ST A GE 2-D D WT 2.1 Tw o Priliminary Devices Used in the Arc hitecture for Rearrangemen t 2.1 Tw o Priliminary Devices Used in the Arc hitecture for Rearrangemen t Lu parent B D A C D B A C LH LH HH HH HL HL 1 1 1 1 1 1 1 HL1 LH2 HH2 Lr1 Hr1 LL3 HL2 HL2 LH2 HH2 LH3 HH3 h(k )LLm-1 1 1 r g(k )LLm- (n ,2n -k ) 1 1 1 k1 k1 1 k2 2 2 2 2 2 k2 k2 k2 c c c c 2 2 column-major high/low-pass filtering th stage separable 2-D DWT formula and the corresponding simplied expressions m 1 2 m 1 2 2 1 1 HHm(n , n )= 2 m m m m m m h(k )Hr (2n -k , n ) HLm Hr 1 2 m m m m 2 2 h(k ) Lr (2n -k , n ) LLm Lr Hr LLm- 1 1 2 1 g(k )Hr (2n -k , n ) HHm Hr 2 1 (n , 2n -k ) Lr LLm- 2 1 r A1 A3 C1 C3 A2 A4 C2 C4 B1 B3 D1 D3 B2 B4 D2 D4 grandparent Lr (n , n )= input image L r1 H r1 LH1 HH 1 LL1 HL 1 LH HH 1 1 HL1 L r2 H r2 LH HH 1 HL 1 LH 2 HH1 LL2 HL 2 filtering high/low -pass row-major filtering high/low-pass column-major filtering high/low -pass row-major filtering high/low-pass column-major filtering high/low -pass row-major (a) the ralation of parent-children in two-stage DWT (b) the relation of parent-children in three -stage DWT (h,g are low-pass and high-pass filter respectively) (c) the m 1 LHm (n ,n )= 2 LLm (n , n )= 1 2 Hr (n , n )= HLm (n , n )= 2 g(k )Lr (2n -k , n ) LHm Lr 1 column-major high/low -pass filtering the corresponding simplified expressions of the formula row-major high/low -pass filtering the corresponding simplified expressions of the formula (d) the three-stage separable 2-D DWT Fig. 1. the algorithms of EZW and 2-D D WT D B A C A1 A3 C1 C3 A2 A4 C2 C4 B1 B3 D1 D3 B2 B4 D2 D4 grandparent parent B D A C parent B D A C Fig. 1. the algorithms of EZW and 2-D D WT N w is the width of the w a v elet lter and N is the width or the heigh t of the input (square) image to D WT. 2.1 Tw o Priliminary Devices Used in the Arc hitecture for Rearrangemen t 2.1 Tw o Priliminary Devices Used in the Arc hitecture for Rearrangemen t (1)The Pro cessing Unit(PU) sho wn as in Figure 2 rearranges the calculation of w a v elet ltering so that the lter is cut to half taps based on the symmetry b et w een the negativ e and p ositiv e w a v elet lter co eÆcien ts. x, a and c are the input sequence, lo w- and high-pass ltering output sequence resp ectiv ely . While a datum of sequence x is fed and shifted in to the PU p er clo c k cycle, a datum of a is calculated ev ery ev en clo c k cycle and a datum of c is calculated ev ery o dd clo c k cycle. The PU in Figure 2(a) can b e extended to a parallel format as in Figure 2(b) where if a n um b er of data from sequence x x k +8 , x k +7 , ..., x k are fed to the PU in parallel at a clo c k cycle, then x k +9 , x k +8 , ..., x k +1 are fed at the next cycle. (2)In Figure 3 the TU is a systolic arra y with (N w +3)N cells, where 470 D. Peng and M. 2.1 Tw o Priliminary Devices Used in the Arc hitecture for Rearrangemen t N is h undreds or thousands of times greater than N w for most applications of 2-D w a v elet transforms (e.g. image/video systems). A cell transfers its con ten t to the next adjacen t cell once it receiv es a datum from its preceding cell. The leftmost cells in o dd ro ws and righ tmost cells in ev en ro ws ha v e output p orts and cop y their data to outside. The upp er-left cell has an input p ort and the TU uses it to reciev e the input sequence. An elemen t in matrix X is fed to the TU p er clo c k cycle in the order according to the indices in Figure 3(b). The TU's (N w +3) outputs and its newly arriv ed elemen t b elong to the same column in X. An example for the p ositions of the X's elemen ts in TU after 3N clo c k cyles of inputting X is illustrated in Figure 3(c). 2.2 the Prop osed Arc hitecture and the Analysis of Its Op erations the structures of PU (Pro cessing Unit) (h4, 0) (h3, h4) C0 C1 (h0, g1) C4 C3 (h1, g2) (h2, g3) C2 a9 a8 a 5 0 A D D E R (h, g) Xi/Yi Pi qi a6 Xi=(Pi+qi)h (even clock cycle), Yi=(Pi+qi)g (odd clock cycle), a4 a7 a 2 a1 a3 (b) parallel filter D D D D (a) systolic filter A D D E R C i (h, g) Xi/Yi Pi qi Pi-1 C0 C1 C2 C3 C4 (h4,0) (h3, g4) (h2, g3) (h1, g2) (h0, g1) 0 a a 2 1 Xi=(Pi+qi)h (even clock cycle), Yi=(Pi+qi)g (odd clock cycle), Pi-1=Pi Fig. 2. the structures of PU (Pro cessing Unit) (h4, 0) (h3, h4) C0 C1 (h0, g1) C4 C3 (h1, g2) (h2, g3) C2 a9 a8 a 5 0 A D D E R a6 a4 a7 a 2 a1 a3 k cycle) D D D D (a) systolic filter A D D E R C0 C1 C2 C3 C4 (h4,0) (h3, g4) (h2, g3) (h1, g2) (h0, g1) 0 ....a a 2 1 Xi=(Pi+q Xi/Yi Fig. 2. the structures of PU (Pro cessing Unit) INPUT Y Y Y Y Y ....... ........ ....... ........ ....... ........ ....... ........ ....... ........ ....... ........ ....... ........ Nw+3 rows , N columns regular row-major indexing of matrix X snake-like row-major indexing of matrix X ... ... 1 2 N N+1 N+2 2N 2N+1 2N+2 3N ... ... ... ... ... ... ... ... ... ... ... (a) (b) (c) 1 N-1 N N+1 N+2 2N 2N+1 3N 3N-1 2 ................ ................ 1 3 Nw+3 Nw+2 Nw+1 Y Fig. 3. A new transp ose unit (TU) snake-like row-major indexing of matrix X ... ... ... ... ... ... 1 N-1 N N+1 N+2 2N 2N+1 3N 3N-1 Nw+3 rows , N columns (a) (c) Fig. 3. A new transp ose unit (TU) Due to the ro w-ma jor dy adic subsampling, the ro w-ma jor high/lo w-pass l- tering is alternativ ely executed b y the PU 1 in Figure 4 p oin t b y p oin t in eac h ro w. Based on similar column-ma jor dy adic subsampling, the PU 2 tak es turns to execute column-ma jor high/lo w-pass ltering and selects appropriate inputs from TU 1 to generate four sibling co eÆcien ts consecutiv ely . 2.2 the Prop osed Arc hitecture and the Analysis of Its Op erations The arc hitecture for the rearrangemen t of D WT is prop osed in Figure 4, and the corresp onding timing of op erations is presen ted in Figure 5. Ev ery four sibling co eÆcien ts in the rst decomp osition stage are designed to b e calculated together (mean while their paren t is generated b y PU 3 ). Non-Memory-Based and Real-Time Zerotree Building 471 (h4, 0) (h3, h4) C0 C1 (h0, g1) C4 C3 (h1, g2) (h2, g3) C2 a9 a8 a 5 0 A D D E R (h, g) Xi/Yi Pi qi a6 Xi=(Pi+qi)h (even clock cycle), Yi=(Pi+qi)g (odd clock a4 a7 a 2 a1 a3 (b) parallel filter D D D D (a) systolic filter A D D E R C i (h, g) Xi/Yi Pi qi Pi-1 C0 C1 C2 C3 C4 (h4,0) (h3, g4) (h2, g3) (h1, g2) (h0, g1) 0 ..a a 2 1 Xi=(Pi+qi)h (even clock cycle), Yi=(Pi+qi)g (odd clock cycle), Pi-1=Pi Fig. 2. the structures of PU (Pro cessing Unit) INPUT Y Y Y Y Y ....... ........ ....... ........ ....... ........ ....... ........ ....... ........ ....... ........ ....... ........ Nw+3 rows , N columns regular row-major indexing of matrix X snake-like row-major indexing of matrix X ... ... 1 2 N N+1 N+2 2N 2N+1 2N+2 3N ... ... ... ... ... ... ... ... ... ... ... (a) (b) (c) 1 N-1 N N+1 N+2 2N 2N+1 3N 3N-1 2 ................ ................ 1 3 Nw+3 Nw+2 Nw+1 Y Fig. 3. A new transp ose unit (TU) 471 Non Memory Based and Real Time Zerotree Building (h4, 0) (h3, h4) C0 C1 (h0, g1) C4 C3 (h1, g2) (h2, g3) C2 a9 a8 a 5 0 A D D E R (h, g) Xi/Yi Pi qi a6 Xi=(Pi+qi)h (even clock cycle), Yi=(Pi+qi)g (odd clock cycle a4 a7 a 2 a1 a3 (b) parallel filter D D D D (a) systolic filter A D D E R C i (h, g) Xi/Yi Pi qi Pi-1 C0 C1 C2 C3 C4 (h4,0) (h3, g4) (h2, g3) (h1, g2) (h0, g1) 0 .a a 2 1 Xi=(Pi+qi)h (even clock cycle), Yi=(Pi+qi)g (odd clock cycle), Pi-1=Pi Fig. 2. 3 THE DESIGN EXTENDED TO GENERAL ST A GES OF D WT No w w e consider what should b e done to mo dify the arc hitecture in Figure 4 for m stages of w a v elet decomp osition. Because the input image is fed in to the system in the same w a y as b efore, the rst stage ro w-ma jor high/lo w-pass ltering is still p erformed in PU 1 alternativ ely as designated in Figure 5. Regarding the rst stage column-ma jor high/lo w-pass tering p erformed in PU 2 , w e note that there are 4 m1 co eÆcien ts in the rst stage decomp osition corresp onding to the same ancestor in the last stage (stage m) decomp osition. T o satisfy the restriction of generating paren t and c hildren sim ultaneously , it is re- quired that these 4 m1 \kindred" co eÆcien ts b e calculated together. (Mean while these co eÆcien ts' paren ts in the in termediate stages of decomp ostion should b e calculated together to o.) Note that these 4 m1 co eÆcien ts are lo cated in 2 m1 adjacen t ro ws and 2 m1 adjacen t columns in their subband. PU 2 should al- ternativ ely select appropriate inputs among 2 m1 dieren t groups of parallel column-ma jor data from TU 1 , and p erform column-ma jor ltering to generate the 4 m1 kindred co eÆcien ts in turns, where the co eÆcein ts calculated with the same group of input b elong to the same ro w. Accordingly , TU 1 is an extended v ersion in Figure 3(a) and is supp osed to ha v e output p orts Y 1 , ..., Y N w +M with M equal to 2 m . PU 3 carries out the rest computation in D WT. The second stage decomp o- sition is ac hiev ed as follo ws. In the rst quarter of the p erio d when 2 m ro ws of input image are fed to the system, PU 3 gets its inputs, i.e., the co eÆcien ts in LL 1 subband from TU 1 , and alternativ ely p erforms the second stage lo w/high- pass ro w-ma jor con v olution. 2.2 the Prop osed Arc hitecture and the Analysis of Its Op erations ) PU3 is the hybrid version of PU that can take either sequential or parallel inputs. ) Feedback block consists of 2 separate TUs and Demultriplexers to select Lr2 / Hr2 into respective TU and to select outputs from 2 TUs into PU3. Input Image 472 D. Peng and M. Lu PU1 TU1 Y1 Y DM 1 PU2 4q 4q+1 4q+2 4q+3 LL1 LH HH HL 1 1 1 Nw-point column-wise data .......... ..... HH HL2 LH2 LL2 3 PU ........ Feedback block Lr / Hr 2 2 Nw+3 Input Image lexer that select Nw-point data from Nw+3 outputs of TU1. of 2 separate TUs and Demultriplexers to select Lr2 / Hr2 into respective TU and to select outputs from 2 TUs into PU3 Fig. 4. The arc hitecture for t w o-stage D WT and the zerotree building 2.2 the Prop osed Arc hitecture and the Analysis of Its Op erations The order to cal- culate the siblings is as A, then B, C and D in the example of four siblings illustrated in Figure 1(a). After PU 2 's calculation of p oin t A b y taking Y 1 , ..., Y N w as inputs, PU 2 has to calculate B b y taking Y 3 , ..., Y N w +2 as inputs, then PU 2 comes bac k to tak e Y 1 , ..., Y N w to calculate C, then PU 2 tak es Y 3 , ..., Y N w +2 again to calculate D. In Figure 5 it can b e seen that PU 2 calculates the same kind of column- ma jor con v olution (high-pass or lo w-pass ltering) during the p erio d when a ro w of input image is sequen tially fed to the system in N clo c k cycles, and the co eÆcien ts in four subbands LL 1 , LH 1 , HL 1 and HH 1 are calculated in turns in a longer p erio d when four ro ws of input image are fed. The righ t column in Figure 5 is to sho w the op erations of PU 3 in Figure 4. In PU 3 the second stage of D WT is completed and the paren t co eÆcien ts are generated. PU 3 tak es sequen tial input of LL 1 co eÆcien ts from PU 2 to p erform ro w-ma jor ltering in the rst quarter of the p erio d during whic h four ro ws of input image are fed to the system. Then in the next three quarters (i.e., i=4q+1, 4q+2 or 4q+3 in Figure 5), PU 3 p erforms the column-ma jor con v olutions b y taking the result from the rst quarter's ro w-ma jor ltering as input. D. Peng and M. Lu 472 PU1 TU1 Y1 Y DM 1 PU2 4q 4q+1 4q+2 4q+3 LL1 LH HH HL 1 1 1 Nw-point column-wise data .......... ..... HH2 HL2 LH2 LL2 3 PU ........ Feedback block Lr / Hr 2 2 Nw+3 1) DM1 is a demultiplexer that select Nw-point data from Nw+3 outputs of TU1. 2) PU2 is a paralle filter as in Figure 2(b) and has four output ports active at different time. 4 PERF ORMANCE ANAL YSIS AND CONCLUSION Since N w (the width of w a v elet lters) is far less than N (the width or length of input image) and the size of b oudary eect of w a v elet transforms is only dep enden t on N w , in this pap er w e ignore the b oudary eect to simplify our expressions kno wing that it can b e resolv ed b y a little adjustmen t in either timing or arc hitecture. The area of the prop osed arc hitecture is dominated b y PUs and TUs. A PU con tains pN w MA Cs (Multiply er and Accum ulator Cell), where p is the n um b er of precision bits of data, th us three PUs con tain 3pN w MA Cs. Because a TU is necessary for the column-ma jor ltering in ev ery stage decomp osition, and the n um b er of cells in TU at the i th stage decomp osition is (N/2 i )(N w +2 i ), where the rst item is the length of a ro w and the second item is the n um b er of ro ws in the TU, the total area for TUs is O(mN+N w N), where m is the n um b er of stages in D WT. Note that the TUs except TU 1 are hidden in PU 3 's feedbac k blo c k. Th us the whole area of the arc hitecture for m stage D WT is A=O(pNN w +pNm). The input image is assumed to b e fed with one pixel p er clcok cycle. The system's latency (execution time) T is N 2 clo c k cycles. Th us the pro duct of A and T for the system is O(pN 3 (N w +m)), where N 2 is the input size of the algorithm. Our prop osed arc hitecture is comparable to con v en tional D WT arc hitectures ([10]-[14]) in the asp ect of area, latency , the pro duct of them, or the hardw are utilization, ev en though not only the D WT but also the zerotree building is ac hiev ed in this arc hitecture. 1. J.M. Shapiro, Em b edded image co ding using zerotrees of w a v elet co eÆcien ts, IEEE T r ansactions on Signal Pr o c essing, V olume: 41, 1993, P age(s): 3445 -3462. 4 PERF ORMANCE ANAL YSIS AND CONCLUSION W e ha v e prop osed a non-memory-based design in whic h the input image is recursiv ely decomp osed b y D WT and zerotrees are built in real-time. The compu- tation of w a v elet-based zerotree co ding is strongly featured b y the computation lo calit y in that the calculations of co eÆcien ts on a certain zerotree (only) dep end on the same lo cal sub-area of the 2-D inputs. This desirable feature has b een exploited in this pap er b y calculating c hildren and their paren t sim ultaneously in the rearranged D WT, so that most in termediate data need not b e held for future calculations. 3 THE DESIGN EXTENDED TO GENERAL ST A GES OF D WT The calculated results, or the co eÆcien ts in Lr 2 and Hr 2 are stored in t w o TUs hidden in PU 3 's feedbac k blo c k. In the second quarter, the Lr 2 co eÆcien ts are fed bac k to PU 3 to b e column-ma jor ltered to get the results in LL 2 and LH 2 . In the third and fourth quarter, the Hr 2 p oin ts Non-Memory-Based and Real-Time Zerotree Building 473 are fed bac k to PU 3 to b e used to calculate out HL 2 and HH 2 resp ectiv ely . The PU 3 ac hiev es further stage decomp ositions in the a v ailable in terv als during its execution of the second stage decomp osition. By reason of limit space in this pap er, the op erations of pro cessors are de- scrib ed with basic principles and not with man y details. T o sum up, TU 1 is mo died to ha v e (N w +M) ro ws; PU3's feedbac k blo c k has c hanged a little big- ger so that it holds N w +M ro ws of co eÆcien ts in the results of ro w-ma jor w a v elet ltering; and the switc hes ha v e b ecome complicated to select appropriate data at diren t time. References 1. J.M. Shapiro, Em b edded image co ding using zerotrees of w a v elet co eÆcien ts, IEEE T r ansactions on Signal Pr o c essing, V olume: 41, 1993, P age(s): 3445 -3462. D. Peng and M. Lu 474 2. A. Said, W.A. P earlman, A new, fast, and eÆcien t image co dec based on set partitioning in hierarc hical trees, IEEE T r ansactions on Cir cuits and Systems for Vide o T e chnolo gy, V olume: 6, June 1996, P age(s): 243 -250. 3. Zixiang Xiong, K. Ramc handran, M.T. Orc hard, W a v elet pac k et image co ding using space-frequency quan tization, IEEE T r ansactions on Image Pr o c essing, V ol- ume: 7, June 1998, P age(s): 892 -898. 4. Zixiang Xiong, K. Ramc handran, M.T. Orc hard, Space-frequency quan tization for w a v elet image co ding, IEEE T r ansactions on Image Pr o c essing, V olume: 6, Ma y 1997, P age(s): 677 -693. , g ( ) 5. M. V etterli, J. Ko v acevic, W a v elets and Subband Co ding, Pr entic e Hal l, 1995. 6. Li-Minn Ang, Hon Nin Cheung, K. Eshraghian, VLSI arc hitecture for signicance map co ding of em b edded zerotree w a v elet co eÆcien ts, Pr o c e ddings of 1998 IEEE Asia-Pacic Confer enc e Cir cuits and Systems, 1998. P age(s): 627 -630. 7. Jongw o o Bae, V. K. Prasanna, A fast and area-eÆcien t VLSI arc hitecture for em b edded image co ding, Pr o c e e dings of International Confer enc e on Image Pr o- c essing, V olume: 3, 1995, P age(s): 452 -455. 8. J.M. Shapiro, A fast tec hnique for iden tifying zerotrees in the EZW algorithm, Pr o- c e e dings of 1996 IEEE International Confer enc e on A c oustics, Sp e e ch, and Signal Pr o c essing, V olume: 3, 1996, P age(s): 1455-1458. 9. J. V ega-Pineda, M. A. Suriano, V. M. Villalv a, S. D. Cabrera, Y.-C. Chang, A VLSI arra y pro cessor with em b edded scalabilit y for hierarc hical image compression,1996 IEEE International Symp osium on Cir cuits and Systems, V olume: 4, 1996, P age(s): 168 -171. 10. References A is from Yi,.......Y (see TU’s output in figure 3) the A is from Yi+1,....Y , A is from Yi+2k+1,.........Y k+ Nw if i=4q+3 1 2 3 r k c are on the right side of "r" arrow, and A is fromthe ith row,then A (1).The meaning of superscript "k" is explained as the following.(k is an integer) Using the simplified expression as in figure 1(c),we call the calculation of as "r" arrow; as "c" arrow. r k c Suppose A corresponds to a part of a component in figure 1 (d). If row-wise signal A and A k is from the kth row. If column-wise signal A and A k Nw+i are on the right side of "c" arrow, and A is from Yi,.....Y (See TU’s output in figure 3), then A k Nw+2k is from Yi+2k,......Y (in the same column) k are on the right side of "r" arrow, and A is fromthe ith row,then A If A and A are on the left side of "c" arrow or "r" arrow,they are corresponding to 2 coefficients inthe same column and in ith row and (i+k)th row respectively. The meaning of "+" is explained as the following: k k The meaning of + is explained as the following: (they are in the same column) + k+ Nw+i k+ Nw+i+1 Column-wise signal A ,A and A are on the right side of "c" arrow. A is from Yi,.......Y (see TU’s output in figure 3) the A is from Yi+1,....Y , A is from Yi+2k+1,.........Y k+ Nw+i+2k+1 (2) Because of PU’s feature of alternative high/low -pass filtering,we use A and A alternative to get column-wise low-high pass outputs. + e reason why we use A and A 1 ( A standing for Lr , LL 1 1 1 C C 1 ) alternatively to generate B and B ( B standing for LL , LH ,.....) is based on the restriction that any siblings be gene 1 1 1 C consecutively. Because of dyadic downsamplings for column-wise convojution. B A if B A C 1 1 C consecutively. Because of dyadic downsamplings for column-wise convojution. B A if B A C (4) Lr and Hr are fed in 2 seperate TUs in PU3’s feedback block. The TUs can hold Nw+2 rows of data at most. References Jer Min Jou, P ei-Yin Chen, Y eu-Horng Shiau, Ming-Shiang Liang, A scalable pip elined arc hitecture for separable 2-D discrete w a v elet transform, Design A u- tomation Confer enc e, Pr o c e e dings of the ASP-D A C '99. Asia and South Pacic, V olume: 1, P age(s): 205 -208. 11. M. Vish w anath, R. M. Ow ens, M. J. Irwin, VLSI arc hitectures for the discrete w a v elet transform, IEEE T r ansactions on Cir cuits and Systems II: A nalo g and Digital Signal Pr o c essing, V olume: 42, Ma y 1995, P age(s): 305 -316. 12. Ch u Y u, Sao-Jie Chen, Design of an eÆcien t VLSI arc hitecture for 2-D discrete w a v elet transforms, IEEE T r ansactions on Consumer Ele ctr onics, V olume: 45, F eb. 1999, Page(s): 135 -140. 13. V. Sundarara jan, K. K. P arhi, Syn thesis of folded, pip elined arc hitectures for m ulti- dimensional m ultirate systems, Pr o c e e dings of the 1998 IEEE International Con- fer enc e on A c oustics, Sp e e ch and Signal Pr o c essing, V olume: 5, 1998, P age(s): 3089 -3092. 14. Ch u Y u, Sao-Jie Chen, VLSI implemen tation of 2-D discrete w a v elet transform for real-time video signal pro cessing, IEEE T r ansactions on Consumer Ele ctr onics, V olume: 43, No v. 1997, P age(s): 1270-1279. 15. T. Ac hary a, P o-Y ueh Chen, VLSI implemen tation of a D WT arc hitecture, Pr o c e e d- ings of the 1998 IEEE International Symp osium on Cir cuits and Systems, V olume: 2, 1998, P age(s): 272 -275. 475 Non-Memory-Based and Real-Time Zerotree Building real-time Alg. References DWT amenable to EZW { for i=0 to N-1 / * row / for j=0 to N-1 / column / Do DWT(i,j) } DWT(i,j) / q,s are any non-negative integers */ { if i=4q what PU does what PU does what PU does if j=even Lr x LL Lr if j=odd Lr x LL Lr if j=4s Lr LL if j=4s+1 Hr LL if j=4s+2 Lr LL if j=4s+3 Hr LL if i=4q+1 if i=4q+2 if j=even Lr x LH Lr if j=odd Lr x LH Lr if j=4s LL Lr if j=4s+2 LH Lr if j=even Lr x HL Hr if j=odd Hr x HL Hr if j=even Lr x HH Hr if j=odd Hr x HH Hr if j=4s+2 HH Hr 1 1 1 1 1 1 1 1 1 2 2 2 1 1 1 1 1 1 1 1 1 1 1 if j=4s+2 HL Hr 1 1 1 + 1+ 1 1 1 2 2 2 2 2 2 2 2 1 1 1 1 2 + + 1 1 1 1 1 1 1 1+ 1 + r r r r r r r r r r r r c c c c c c c c c c c c Notation: (1).The meaning of superscript "k" is explained as the following.(k is an integer) Using the simplified expression as in figure 1(c),we call the calculation of as "r" arrow; as "c" arrow. Suppose A corresponds to a part of a component in figure 1 (d). If row-wise signal A and A k is from the kth row. If column-wise signal A and A k Nw+i are on the right side of "c" arrow, and A is from Yi,.....Y (See TU’s output in figure 3), then A k Nw+2k is from Yi+2k,......Y (in the same column) If A and A are on the left side of "c" arrow or "r" arrow,they are corresponding to 2 coefficients inthe same column and in ith row and (i+k)th row respectively. The meaning of "+" is explained as the following: (they are in the same column) + k+ k Nw+i k+ Nw+i+1 Column-wise signal A ,A and A are on the right side of "c" arrow. References Careful readers may find that LL Lr and LH Lr are executed alternatively point by point, however, HL Hr and HH Hr are executed alternatively row-by row. This paradox can be resolved by a little manipulation in 2 TUs in feedback block . Anyway, during the time of 4q+1<=i<=4q+3, no new data are generated for Lr or Hr ,so the old data in feedback block can be held until i=4q+4. (5) This real time algorithm dictates the operations happening in figure 4 The control signals for PU TU DM PU PU feedback block can be easily implemented locally and periodically Their details are not 2 2 2 2 2 2 2 2 2 2 2 2 c c c c 1 1 1 2 (4) Lr and Hr are fed in 2 seperate TUs in PU3’s feedback block. The TUs can hold Nw+2 rows of data at most. Careful readers may find that LL Lr and LH point by point, however, HL Hr and HH Hr are executed alternatively row-by row. This paradox can be resolved by a little manipulation in A d i th ti f 4 +1< i< 4 +3 d t t d f L H th ld d t i f db k bl k b h ld til i 4 +4 2 2 2 2 2 2 2 2 2 c c Anyway, during the time of 4q+1<=i<=4q+3, no new data are generated for Lr or Hr ,so the old data in feedback block can be held until i=4q+4. 2 2 2 2 ) This real time algorithm dictates the operations happening in figure 4. The control signals for PU ,TU ,DM ,PU ,PU , feedback block can be easily implemented locally and periodically. Their details are not discussed because of the limited length of this paper. 1 1 1 2 3 Fig. 5. The timing for the op erations in the arc hitecture for t w o-stage D WT and the zerotree building Fig. 5. The timing for the op erations in the arc hitecture for t w o-stage D WT and the zerotree building
https://openalex.org/W2598662297	https://scholarworks.wm.edu/cgi/viewcontent.cgi?article=1773&context=vimsarticles	English	null	Ghost of invasion past: legacy effects on community disassembly following eradication of an invasive ecosystem engineer	Ecosphere	2,017	cc-by	10,665	Follow this and additional works at: https://scholarworks.wm.edu/vimsarticles Follow this and additional works at: https://scholarworks.wm.edu/vimsarticles Part of the Aquaculture and Fisheries Commons W&M ScholarWorks W&M ScholarWorks This article is available at W&M ScholarWorks: https://scholarworks.wm.edu/vimsarticles/773 Authors Authors PL Reynolds, J Glanz, S Yang, C Hann, J Couture, and E Grosholz Recommended Citation Recommended Citation Reynolds, PL; Glanz, J; Yang, S; Hann, C; Couture, J; and Grosholz, E, "Ghost of invasion past: legacy effects on community disassembly following eradication of an invasive ecosystem engineer" (2017). VIMS Articles. 773. https://scholarworks.wm.edu/vimsarticles/773 This Article is brought to you for free and open access by W&M ScholarWorks. It has been accepted for inclusion in VIMS Articles by an authorized administrator of W&M ScholarWorks. For more information, please contact scholarworks@wm.edu. Ghost of invasion past: legacy effects on community disassembly following eradication of an invasive ecosystem engineer PAMELA L. REYNOLDS,1,2, JESS GLANZ,1 SYLVIAYANG,1,3 COURTNEY HANN,1,4 JESSICA COUTURE,1,5 AND EDWIN GROSHOLZ1 PAMELA L. REYNOLDS,1,2, JESS GLANZ,1 SYLVIAYANG,1,3 COURTNEY HANN,1,4 JESSICA COUTURE,1,5 AND EDWIN GROSHOLZ1 1Department of Environmental Science and Policy, University of California Davis, Davis, California 95616 USA 2Virginia Institute of Marine Science, Gloucester Point, Virginia 23062 USA 3Shannon Point Marine Center, Western Washington University, Anacortes, Washington 98221 USA 4College of Earth, Ocean and Atmospheric Sciences, Oregon State University, Corvallis, Oregon 97333 USA 5Bren School of Environmental Science & Management, University of California Santa Barbara, Santa Barbara, California 93106 USA 1Department of Environmental Science and Policy, University of California Davis, Davis, California 95616 USA 2Virginia Institute of Marine Science, Gloucester Point, Virginia 23062 USA 3Shannon Point Marine Center, Western Washington University, Anacortes, Washington 98221 USA 4College of Earth, Ocean and Atmospheric Sciences, Oregon State University, Corvallis, Oregon 97333 USA 5Bren School of Environmental Science & Management, University of California Santa Barbara, Santa Barbara, California 93106 USA Citation: Reynolds, P. L., J. Glanz, S. Yang, C. Hann, J. Couture, and E. Grosholz. 2017. Ghost of invasion past: legacy effects on community disassembly following eradication of an invasive ecosystem engineer. Ecosphere 8(3):e01711. 10.1002/ecs2.1711 Abstract. By changing ecosystem processes and altering the physical landscape, invasive ecosystem engineers can have substantial impacts on ecosystem functions and human economies and may facilitate other non-native species. Eradication programs in terrestrial and aquatic systems aim to reverse the impacts of invasive species and return the system to its pre-invasion conditions. Despite an extensive focus on the impacts of both native and non-native ecosystem engineers, the consequences of removing invasive ecosystem engineers, particularly in coastal ecosystems, are largely unknown. In this study, we quantiﬁed changes in a benthic community following the eradication of the invasive ecosystem engineer, hybrid cord- grass Spartina, in San Francisco Bay, California. We used ﬁeld experimental manipulations to test for persis- tent effects of both aboveground and belowground structural modiﬁcations of the invasive plant on the benthic community. We found signiﬁcant effects of the invasive plant more than four years following eradi- cation. Experimental modiﬁcation of the above- vs. belowground structure of this ecosystem engineer revealed taxonomic speciﬁc effects resulting in hysteresis in the recovery of the benthic food webs. We found that these “legacy effects” resulted from two speciﬁc mechanisms: (1) delayed breakdown of below- ground structures (stems, roots) and (2) persistence of other invasive species whose invasion was facilitated by the ecosystem engineer. March 2017 ❖Volume 8(3) ❖Article e01711 Received 21 December 2016; accepted 3 January 2017. Corresponding Editor: Debra P. C. Peters. Copyright: © 2017 Reynolds et al. 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. E-mail: plreynolds@ucdavis.edu INTRODUCTION The impacts of biological invasions on ecosys- tem functions and human economies are increas- ingly well documented (Simberloff et al. 2013). Ecologists now recognize that the invaders of greatest concern include “ecosystem engineers,” which can alter fundamental ecosystem processes through their impact on the abiotic environment and consequently inﬂuence the distribution and functioning of entire communities (Wright and Jones 2006). By altering the physio-chemical land- scape, invasive ecosystem engineers may in some cases facilitate the invasion of other non-native species (Simberloff and Von Holle 1999, Grosholz 2005, Mendez et al. 2015, Gallardo et al. 2016). These invasion impacts may be reversed by man- agement actions that attempt to completely eradi- cate, or substantially reduce, non-native invasive ecosystem engineers. By reversing the invasion impacts, this could potentially allow the system to return to pre-invasion conditions. However, unin- tended consequences can accompany invader removal, and successful restoration is not a cer- tainty (Zavaleta et al. 2001). These primarily terrestrial studies highlight several mechanisms by which legacy effects of invasive ecosystem engineers may have a persis- tent, long-term inﬂuence on community assem- bly and ecosystem recovery processes and pose signiﬁcant challenges for restoring ecosystem functions and services. However, in contrast to terrestrial ecosystems, we know far less about the consequences of removing non-native ecosys- tem engineers in coastal communities due in large part to the small number of successful removals in these systems (Hacker and Dethier 2009). Some of the consequences of losses of native marine ecosystem engineers through over- exploitation are known (Coleman and Williams 2002), but whether these apply to eradications of invasive engineers is unclear. y Despite a substantial body of work on the changes produced by ecosystem engineers both native and non-native (Crooks 2002, Wright and Jones 2006), we know considerably less about what happens when these engineers are removed (Coleman and Williams 2002, Hacker and Dethier 2009, Corbin and D’Antonio 2012), although there are some compelling examples from terrestrial sys- tems. Studies of invasive grasses showed that increased nutrient and light availability following local eradication increased seedling recruitment and growth of native shrubs (D’Antonio et al. 1998). Other studies quantifying the effects of non- native grasses as secondary invaders following the removal of non-native grazers found that invasive grasses altered light and soil conditions and slo- wed the recovery of native shrubs (Yelenik and Levine 2011). Ghost of invasion past: legacy effects on community disassembly following eradication of an invasive ecosystem engineer PAMELA L. REYNOLDS,1,2, JESS GLANZ,1 SYLVIAYANG,1,3 COURTNEY HANN,1,4 JESSICA COUTURE,1,5 AND EDWIN GROSHOLZ1 Both of these mechanisms are likely to occur in similar systems where below- ground structures breakdown more slowly or where other associated long-lived invaders persist. Our work is among the ﬁrst to quantify the slow rate of change in food web and community processes and the persistent legacy effects of an invasive ecosystem engineer in a coastal ecosystem. We suggest that this delayed transition to pre-invasion conditions could resemble an alternate state that would be misidentiﬁed without a sufﬁcient monitoring interval or recovery duration, with consequences for future management and restoration activity planning. Key words: community disassembly; diversity; ecosystem engineer; epifauna; eradication; infauna; invasive species; restoration; salt marsh; Spartina; structural effects. Received 21 December 2016; accepted 3 January 2017. Corresponding Editor: Debra P. C. Peters. Received 21 December 2016; accepted 3 January 2017. Corresponding Editor: Debra P. C. Peters. Copyright: © 2017 Reynolds et al. 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. E-mail: plreynolds@ucdavis.edu ❖www.esajournals.org March 2017 ❖Volume 8(3) ❖Article e01711 1 1 REYNOLDS ET AL. addition of allelopathic chemicals (Ridenour and Callaway 2001, Grove et al. 2012), with conse- quences for the subsequent colonization by native or non-native species many years after the engi- neer is removed (Von Holle et al. 2013). For exam- ple, co-occurring non-native plants persisted for more than six years after the experimental removal of the invasive tree Acacia longifolia, which reduced native species via accumulation of leaf litter and light reduction (Marchante et al. 2011). A common feature of many of these studies is that there are often persistent effects of the invasive ecosystem engineer long after its removal, referred to as “legacy effects” (Corbin and D’Antonio 2012). March 2017 ❖Volume 8(3) ❖Article e01711 ❖www.esajournals.org INTRODUCTION 1997), and increased levels of sediment organic matter, porewater sulﬁde, and anoxia (Neira et al. 2006). Additionally, infaunal abundance was generally lower within invaded zones com- pared to uninvaded mudﬂats (Levin et al. 2006). These changes to the physio-chemical environ- ment were strongly associated with dramatic changes in the benthic food webs, as supported by stable isotope tracer studies (Levin et al. 2006). The benthic communities shifted from one dominated by surface-feeding species (including bivalves, amphipods, and cirratulid polychaetes) to a community dominated by subsurface detriti- vores, including capitellid polychaetes and tubiﬁ- cid oligochaetes (Levin et al. 2006, Neira et al. 2006). This shift in the community from surface to subsurface invertebrates has potentially important implications for higher trophic levels as well. p g p These invasion-driven changes began to shift beginning in 2006, when California state agen- cies conducted an extensive eradication program to eliminate hybrid Spartina throughout San Francisco Bay using aerial application of herbi- cide (imazapyr). By 2010, hybrid Spartina was eradicated (or nearly eradicated) at most sites within the Bay and this program continues to date. The eradication efforts left dense mats of decaying belowground plant material, elevated sediment topography, and standing dead above- ground stems. y We undertook this challenge by experimen- tally exploring the consequences of the eradica- tion of invasive hybrid cordgrass Spartina (S. alterniﬂora 9 S. foliosa) from San Francisco Bay, California, United States, and the prospects for recovery of the benthic community to pre- invasion conditions. At the height of its invasion (prior to 2006), this ecosystem engineer had transformed previously unvegetated mudﬂats into thick meadows of dense emergent vegeta- tion that produced large quantities of detritus aboveground and created thick, impenetrable rhizome mats belowground. This invasion had profound impacts on the system resulting in greatly altered invertebrate biomass and diver- sity, food web shifts, changes in sediment geo- chemistry, dramatically reduced light and water ﬂow at the sediment surface, increased accretion of ﬁne sediments, and increased tidal elevation (Levin et al. 2006, Neira et al. 2006, Rosso et al. 2006, Tyler et al. 2007). Experimental manipula- tions of sediments and benthic communities inside and outside the invaded zone (Neira et al. INTRODUCTION Additionally, some invasive plant ecosystem engineers modify the mineralization and retention of nitrogen in ways that may inﬂu- ence soil nitrogen for several years after they have been experimentally removed (Maron and Jefferies 2001, Grove et al. 2015). Other engineering inva- ders may change the chemical composition of soils, either via changes in salinity and pH (D’Antonio et al. 1998) or as a function of the We predict that invasive ecosystem engineers in coastal systems may have substantial legacy effects. Wetland soils are frequently water-logged and anoxic and rates of decomposition may be slow, allowing for an extended legacy of below- ground effects of invasive plants. Also, many ben- thic invertebrates have easily dispersed planktonic life stages as well as gregarious recruitment and, in some cases, long life spans. Thus, species that may be facilitated by invasive plants can poten- tially persist long after the ecosystem engineer has been removed, further delaying the recovery of the community to the pre-invasion condition. y p Studies to date documenting the removal of invasive salt marsh plants have provided unique opportunities to investigate the consequences of eradicating invasive marine engineers, although they have typically focused on the responses of single taxa or speciﬁc sets of associated non-native species (Patten and O’Casey 2007, Holsman et al. March 2017 ❖Volume 8(3) ❖Article e01711 ❖www.esajournals.org 2 REYNOLDS ET AL. REYNOLDS ET AL. 2010). These studies have also provided insights into the effects of partial removal of the invasive engineer (Reeder and Hacker 2004) and potential long-term impacts of invader removal on ero- sional processes and geomorphology (Sheehan and Ellison 2015). However, studies on the magni- tude and the duration of legacy impacts of ecosys- tem engineers on entire benthic communities are needed if we hope to understand the direction and speed of ecosystem recovery and, ultimately, how to manage the restoration of invaded ecosys- tems (Suding et al. 2004, Norton 2009). A few studies have made progress in this direction in salt marshes (Reeder and Hacker 2004) and macroalgal beds (Piazzi and Ceccherelli 2006), but they have not experimentally investigated the mechanisms underlying legacy impacts on the restoration process. Thus, mechanistic experi- ments are critical to determine whether processes producing hysteresis and thwarting recovery after eradication of an invasive ecosystem engineer are similar in coastal ecosystems as well as in other, more well studied systems. ❖www.esajournals.org March 2017 ❖Volume 8(3) ❖Article e01711 INTRODUCTION 2006) showed that these physical effects of hybrid Spartina produced lower levels of sedi- ment chlorophyll-a (i.e., epibenthic microalgae), which is the main primary producer on unvege- tated mudﬂats in this region (Kwak and Zedler g We used this large-scale eradication to exam- ine the possibility of legacy effects of above- and belowground effects of hybrid Spartina. We hypothesized that aboveground legacy effects might include the persistence of long-lived spe- cies initially supported by refugia and other engi- neering modiﬁcations by the invasive plant. Also, the potential for persistent effects on the sediment surface, including sediment character- istics such as elevation, may also result in legacy effects on the abundance and composition of infauna. We further hypothesized that below- ground legacy effects of hybrid Spartina may include delayed breakdown of belowground bio- mass consisting of roots and rhizomes, which would continue to inﬂuence benthic communi- ties and food web structure via persistent effects on sediment characteristics including detritus and porewater chemistry. In order to experimentally quantify the legacy effects of hybrid Spartina on benthic communities March 2017 ❖Volume 8(3) ❖Article e01711 ❖www.esajournals.org March 2017 ❖Volume 8(3) ❖Article e01711 3 REYNOLDS ET AL. and potential constraints on the recovery pro- cess, we experimentally added structural ele- ments that mimicked the structure and biomass of invasive hybrid Spartina. We added above- ground structural mimics to reproduce the phys- ical effects on water ﬂow and light on the sediment, and measured changes in sediment composition and porewater chemistry, as well as the abundance and diversity of the species-rich benthic infaunal and epifaunal communities. We also added hybrid Spartina litter in belowground treatments to support detritivores, thus affecting community assembly and altering trophic inter- actions. We reasoned that these manipulations, by recreating much of the physio-chemical envi- ronment of the recently removed hybrid Spartina, would delay the transition of the recovering invertebrate community and/or potentially shift the community back toward the invaded state. We compared these results with appropriate con- trols to understand how quickly, or even if, the community would return to pre-invasion state. We explored the possibility that this system, like many others, might respond much more slowly to the removal of invasive ecosystem engineers than expected, following a slower-than-expected trajectory toward recovery of the pre-invasion food web. mudﬂat state. While eradication is complete at this site, recovery can only be considered in pro- gress. INTRODUCTION Our experimental manipulations were intended to quantify the degree to which eradica- tion sites are actually on their way to recovery. Structural manipulations 2 Six ﬁeld plots (1 m2) were established in each of eight blocks parallel to the shoreline at approximately +1.5 ft mean lower low water (MLLW) within the eradicated hybrid Spartina zone (hereafter “hybrid zone”) adjacent to the Elsie Roemer Bird Sanctuary on 28 June 2013. Plots within each block in the hybrid zone were randomly assigned to one of six structural manipulation treatments to mimic different types of legacy effects (Appendix S1: Fig. S1) including three aboveground manipulations, two below- ground manipulations, and an unmanipulated control (UC). The purpose of these manipula- tions was to determine to what degree the sys- tem has remained in the invaded state vs. transitioning toward the pre-invasion state. The three aboveground manipulations incl- uded additions of either dowels (D) or shade screens (S) to mimic the physical effects on water ﬂow and light availability at the substra- tum surface, respectively. Because Spartina stems remain partially intact following eradica- tion, their engineering effects may persist for a year or more post-eradication. The dowel (D) addition plots contained 500 evenly placed woo- den stakes emerging 50 cm above the sediment with a 900-cm2 opening in the center for access for faunal and sediment sampling. The dowels were intended to mimic both the reduction in water ﬂow and the shading effects of hybrid Spartina and were based on observed stem densities and light attenuation (Neira et al. 2005, 2006). The shade screens (S) were con- structed with a PVC frame covered in Vexar mesh (0.25 cm2 openings) wrapped in burlap. These were placed approximately 0.3 m above and parallel to the sediment surface and were intended to create similar shading effects as the dowels but with minimal effects on ﬂow. Shade screen controls (Vexar without burlap) were included as the third aboveground treatment, but the results from those plots are not included in this analysis as the screens experienced peri- odic fouling. ❖www.esajournals.org Field site and invasion history Field site and invasion history y We conducted our experimental study of reco- very following the eradication of an invasive ecosystem engineer at a long-term study site, the Elsie Roemer Bird Sanctuary in Alameda, CA (37.752589 N, 122.248755 W; Appendix S1: Fig. S1), where the impacts of the hybrid Spartina invasion were experimentally quantiﬁed over a ﬁve-year period from 2001 to 2005 (Neira et al. 2005, 2007, Levin et al. 2006, Tyler et al. 2007). This site (described in Neira et al. 2006) was ﬁrst invaded by hybrid Spartina nearly 40 yr ago, and the invasion continued to spread through 2007. Beginning in 2008, an extensive eradication pro- gram was undertaken using herbicide, with nearly complete eradication of the hybrid at this location by 2011 and no observable hybrid by 2013 (Invasive Spartina Project Report 2013), making it an ideal location for testing for legacy effects on habitat recovery to the pre-invasion March 2017 ❖Volume 8(3) ❖Article e01711 ❖www.esajournals.org 4 REYNOLDS ET AL. REYNOLDS ET AL. The two belowground manipulations entailed either the addition of dead hybrid Spartina litter (L) to mimic the effects of Spartina on the detrital pool, or aeration (A) of sediments using a hand trowel to assess whether modest hastening of the breakdown of the belowground Spartina root mat accelerated the transition of the benthic com- munities toward the pre-invasion state. The litter addition (L) plots received six bags of 20 g dried hybrid Spartina roots and stems buried 5 cm in a standardized array within the center of the plot. This treatment was intended to mimic the signiﬁ- cant amounts of hybrid Spartina litter typical of invaded sites, which provides additional trophic support for benthic detritivores. The quantity of experimentally added litter is within the normal range of belowground biomass typical for this site (Tyler et al. 2007). The aeration (A) treat- ment, despite efforts to regularly maintain this manipulation, showed no changes from the UCs, indicating that modest aeration is insufﬁcient to inﬂuence sediment nor food web characteristics, and is thus not presented here. sediment accretion by the hybrid Spartina prior to its eradication. These two zones were of equiva- lent tidal height prior to the Spartina invasion (Rosso et al. 2006). Furthermore, intact mudﬂat can be found elsewhere at this site at both of our experimental elevations. Field site and invasion history Plots within each block in the mudﬂat zone were randomly assigned to one of two treatments, either UCs or dowel addi- tions (D). Because this zone was never invaded, it provided an area to test whether any observed aboveground structural effects could arise with- out the persistence of any belowground structure. We did not construct belowground manipulations in the mudﬂat due to technical difﬁculties observed in prior transplant experiments and because it was never invaded and consequently never had any belowground plant biomass. Blocks were sampled prior to experimental manipulation. All plots were sampled during daytime low tides at 90 d (17 September 2013) and 270 d (23 March 2014) after treatment estab- lishment. All structural manipulations were then removed and the plots re-sampled after 30 d to assess recovery. Experimental structures were maintained as necessary throughout the experi- ment and drift algae removed where it accumu- lated around plot marking poles. We assessed the effects of these experimental manipulations on the following features of the benthic habitat. p The above- and belowground manipulations were intended to provide the physical structure needed to mimic the conditions present during the Spartina invasion. If species that had been originally facilitated by the Spartina invasion were still present in the system, we predicted that they would potentially increase in abun- dance in response to these re-created conditions. We hypothesized that Spartina-facilitated infau- nal species would still be present if there were legacy effects of the invasion, such as decompos- ing roots and rhizomes that maintained below- ground conditions similar to the invaded state. We also predicted that adding structure and recreating the conditions typical of the invasion would reduce the abundance of surface-feeding species that were more common prior to the invasion on uninvaded mudﬂats. ❖www.esajournals.org March 2017 ❖Volume 8(3) ❖Article e01711 Responses Infauna.—To assess the effects of experimental manipulations on infaunal abundance and com- munity structure, one core (5 cm diame- ter 9 5 cm deep) was collected in the center of each plot on each sampling date. Cores were ﬁxed in 8% formalin for at least two days, then rinsed for 24 h in deionized water before trans- ferring for storage in 70% ethanol. Rose bengal was added to increase invertebrate detectability. Samples were sieved to 300 lm and all retained invertebrate infauna counted, identiﬁed to spe- cies, and classiﬁed to surface or subsurface deposit feeders or predator/omnivore feeding mode (sensu Levin et al. 2006). Infauna were quantiﬁed from within half of the plots on day 90 (n = 4 for 24 total plots), whereas all plots were surveyed on day 270 and again after 30 d of recovery (n = 8 for 48 total plots). Two additional plots were set up in each of eight neighboring blocks at no lower than +1.0 MLLW elevation within the mudﬂat on 22 July 2013 (hereafter “mudﬂat”). Although these mud- ﬂat plots are approximately 5–10 m from the hybrid zone plots, they had no prior history of hybrid Spartina invasion and thus provide a pre- invasion community control. The slight eleva- tional difference between the mudﬂat and hybrid zones was another legacy effect caused by modest Epifauna.—We assessed epifaunal communities in the UC plots and the dowel aboveground March 2017 ❖Volume 8(3) ❖Article e01711 ❖www.esajournals.org 5 REYNOLDS ET AL. REYNOLDS ET AL. 210 rpm for 5 min. We then weighed the result- ing two size fractions (silt vs. sand). structure plots using pit fall traps and visual cen- suses during each sampling event. Pit fall traps (10 cm dia. 9 15 cm depth) were opened at low tide and all captured invertebrates were cen- sused after 24 h. Visual counts of all mobile epi- fauna were made in three quadrats (0.0625 m2) within ﬁxed locations in the plot. Due to large Ilyanassa obsoleta accumulations, total epifaunal abundance (summed across the three quadrats per plot) was quantiﬁed on day 270 and 30 d post-manipulation removal. Chlorophyll-a.—Responses of benthic microalgae were assessed by collecting small (0.5 cm deep) surface sediment cores in the center of each plot, which were frozen and analyzed within 30 d on a 1700 Shimadzu spectrophotometer for chloro- phyll-a concentration as proxy for microalgae accumulation following Neira et al. (2006). Responses We extracted each core with 6 mL of 90% acetone and then sonicated in a cold water bath for three one- minute intervals with a one-minute break between each interval. Samples were then refrozen, shaken to ensure complete extraction, and 24 h later cen- trifuged for 5 min at 4 rpm before placement in the spectrophotometer, where they were read at 665 and 750 nm. Samples were acidiﬁed with HCL to assess phaeophyton. Samples were cali- brated according to overall sediment dry mass. As a follow-up experiment, to assess the potential refuge of the aboveground structure for the invasive Eastern mudsnail I. obsoleta, we con- ducted a tethering experiment in summer 2014. Five adult Ilyanassa were marked and tethered within each of newly constructed dowel and UC plots within the ﬁrst ﬁve original blocks. An additional ﬁve marked untethered (free) snails were added to each plot to test the snails’ disper- sal. Plots were checked after 48 h and scored for the number of recovered live and dead snails. This assay was conducted twice (June and July) and, as responses were consistent, results were pooled for ease of presentation. Individual snails were excluded if there was evidence that the tether broke or there was a failure of glue adhe- sion (22 out of 800 snails). g y Porewater sulﬁde.—We measured sulﬁdes from porewater samplers installed at 10–12 cm depth. Porewater was sampled 48 h after sampler installation and stored in an antioxidant buffer (NaOH + disodium ethylenediaminetetraacetic acid) on ice. Back in the laboratory, the mV for each standard and sample were recorded using a silver sulﬁde ion-selective electrode and com- pared to standards prepared with the buffer, deoxygenated water, and disodium sulﬁde (see Zhang et al. 1997). Belowground biomass.—To assess changes in belowground biomass of the decaying hybrid Spartina root mat during the experiment, one core (25 cm deep, 5 cm diameter) was collected from the center of each plot during the structure removal. Cores were sieved to 500 lm and all root materials dried and weighed. Cores were not collected from the mudﬂat as personal obser- vations and historical data indicated that there was no belowground biomass and Spartina has never been present in this zone. ❖www.esajournals.org March 2017 ❖Volume 8(3) ❖Article e01711 Statistical analyses l y Data were analyzed in R (v3.1.1) (R Core Team 2015) using a generalized linear model with habi- tat by structural manipulation as ﬁxed factors along with spatial block. Because this was not a fully crossed model, we conducted a priori planned contrasts to test among habitats (e.g., eradicated hybrid vs. mudﬂat) and structural manipulations (e.g., UC vs. dowel across habitats, control vs. dowel and shade or all manipulations in eradicated hybrid, and control vs. dowel in mudﬂat only). We used a quasi-poisson distribu- tion as needed for count data. Plots from which infauna were not preserved adequately for identi- ﬁcation and/or in which signiﬁcant maintenance was necessary within one month of the sampling were excluded from the analyses (day 90: one mudﬂat dowel and one hybrid shade out of 24 total sampled plots; day 270 and recovery day 30: two hybrid dowels, one mudﬂat dowel, and one Sediment organic content.—Organic content was quantiﬁed by assessing the total organic matter and the ratio of silt to sand in the sediment. Two sediment cores (10 cm deep) were collected from the center of each plot, frozen, and then dried at 60°C for 24 h prior to sieving through 500-lm mesh to remove debris. To assess the organic matter in the sediment, cores were combusted at 500°C for 6 h to quantify loss on ignition. To assess silt content within the sediment, sediment from the combusted cores was shaken through a 63-lm sieve on an oscillator (KJ-201BD) at March 2017 ❖Volume 8(3) ❖Article e01711 ❖www.esajournals.org March 2017 ❖Volume 8(3) ❖Article e01711 6 REYNOLDS ET AL. Table 1. A priori planned contrasts of structural manipulations across habitats on the infauna for 90 and 270 d after experimental initiation, and 30 d after experimental removal. Response Estimate SE t-Value Pr(>\|t\|) Total infauna abundance Habitat (mudﬂat vs. hybrid) 1.790; 1.277; 0.373 2.546; 0.317; 0.222 0.707; 4.030; 1.683 0.490; <0.001; 0.101 Habitat ctrl vs. dowel 5.100; 1.557; 0.324 3.764; 0.384; 0.316 1.355; 4.075; 1.023 0.194; <0.001; 0.313 Hybrid ctrl vs. aboveground 1.294; 2.476; 0.897 4.411; 0.609; 0.404 0.293; 4.062; 2.222 0.773; <0.001; 0.033 Mudﬂat ctrl vs. aboveground 6.045; 0.408; 0.150 2.772; 0.193; 0.217 2.180; 2.111; 0.691 0.044; 0.042; 0.494 Hybrid ctrl vs. belowground 0.244; 1.059; 0.319 4.103; 0.612; 0.385 0.060; 1.731; 0.827 0.953; 0.092; 0.414 Annelids Habitat (mudﬂat vs. hybrid) 19.250; 1.062; 0.246 28.468; 0.312; 0.236 0.676; 3.401; 1.043 0.509; 0.002; 0.304 Habitat ctrl vs. Statistical analyses l A priori planned contrasts of structural manipulations across habitats on the infauna for 90 and 270 d after experimental initiation, and 30 d after experimental removal. Note: Bold numbers are signiﬁcant (P < 0.05). hybrid UC out of 48 total sampled plots). The infaunal community was further explored using non-metric multidimensional scaling (NMDS) with the vegan package in R. (Fig. 1A). This pattern occurred across mudﬂat and hybrid habitats for total annelid abundance (Fig. 1B) and within the mudﬂat for small non- decapod crustaceans (i.e., amphipods and tanaids, Fig. 1C, Table 1). After 270 d, total infaunal abun- dance was greater in the mudﬂat where no inva- sive Spartina was ever present (i.e., our pre-hybrid Spartina invasion reference habitat), compared to the hybrid zone where eradication had been con- ducted (Fig. 1E). The presence of aboveground (dowels, shade screen), but not belowground (lit- ter) manipulations promoted total infaunal abun- dance in the hybrid zone (Table 1). Aboveground Statistical analyses l dowel 92.574; 0.531; 0.213 42.083; 0.420; 0.341 2.200; 1.263; 0.623 0.043; 0.215; 0.537 Hybrid ctrl vs. aboveground 90.750; 1.753; 0.703 49.308; 0.604; 0.425 1.840; 2.900; 1.656 0.084; 0.006; 0.106 Hybrid ctrl vs. belowground 67.153; 0.198; 0.073 45.865; 0.244; 0.402 1.464; 0.811; 0.311 0.163; 0.423; 0.758 Mudﬂat ctrl vs. aboveground 46.074; 0.779; 0.230 30.992; 0.591; 0.234 1.487; 1.317; 0.572 0.157; 0.423; 0.571 Polychaetes Habitat (mudﬂat vs. hybrid) 0.100; 1.361; 0.613 1.490; 0.273; 0.304 0.067; 4.980; 2.016 0.947; <0.001; 0.051 Habitat ctrl vs. dowel 5.904; 0.699; 0.277 2.203; 0.355; 0.431 2.680; 1.968; 0.643 0.016; 0.057; 0.525 Hybrid ctrl vs. aboveground 6.328; 1.895; 0.815 2.581; 0.541; 0.580 2.451; 3.503; 1.405 0.026; 0.001; 0.169 Hybrid ctrl vs. belowground 4.507; 0.264; 0.071 2.401; 0.194 0.269 1.877; 1.359; 0.265 0.079; 0.182; 0.792 Mudﬂat ctrl vs. aboveground 3.457; 0.919; 0.234 1.623; 0.532 0.560 2.131; 1.729; 0.418 0.049; 0.093; 0.678 Oligochaetes Habitat (mudﬂat vs. hybrid) 1.981; 0.545; 1.386 1.359; 0.678; 0.747 1.458; 0.804; 1.855 0.164; 0.426; 0.071 Habitat ctrl vs. dowel 0.233; 0.313; 0.098 1.809; 0.959; 1.138 0.129; 0.326; 0.086 0.899; 0.746; 0.932 Hybrid ctrl vs. aboveground 0.109; 1.480; 0.488 1.182; 1.249; 0.836 0.092; 1.186; 0.584 0.928; 0.243; 0.563 Hybrid ctrl vs. belowground 0.286; 0.052; 0.066 1.074; 0.610; 1.025 0.266; 0.086; 0.064 0.794; 0.932; 0.949 Mudﬂat ctrl vs. aboveground 0.639; 0.553; 0.074 1.647; 1.204; 0.774 0.388; 0.460; 0.056 0.703; 0.648; 0.924 Crustaceans Habitat (mudﬂat vs. hybrid) 1.800; 0.890; 0.050 2.546; 1.014; 0.502 0.707; 0.878; 0.099 0.490; 0.386; 0.922 Habitat ctrl vs. dowel 5.100; 5.162; 1.233 3.764; 1.060; 0.719 1.355; 4.870; 1.716 0.194; <0.001; 0.095 Hybrid ctrl vs. aboveground 1.294; 5.133; 2.400 4.411; 1.769; 0.797 0.293; 2.902; 3.040 0.773; 0.006; 0.005 Hybrid ctrl vs. belowground 0.245; 2.554; 0.437 4.103; 0.552; 0.592 0.060; 4.624; 0.738 0.953; <0.001; 0.465 Mudﬂat ctrl vs. aboveground 6.045; 2.149; 0.026 2.772; 1.789; 0.853 2.180; 1.201; 0.031 0.045; 0.237; 0.976 Species richness Habitat (mudﬂat vs. hybrid) 0.026; 4.762; 1.373 0.144; 0.813; 1.381 0.180; 5.861; 0.994 0.859; <0.001; 0.327 Habitat ctrl vs. dowel 0.173; 4.998; 1.008 0.209; 1.216; 2.068 0.826; 4.110; 0.487 0.421; <0.001; 0.629 Hybrid ctrl vs. aboveground 0.263; 11.746; 2.947 0.243; 1.460; 2.482 1.080; 8.046; 1.187 0.296; <0.001; 0.243 Hybrid ctrl vs. belowground 0.095; 0.264; 0.310 0.229; 0.848; 1.441 0.417; 0.311; 0.215 0.682; 0.757; 0.831 Mudﬂat ctrl vs. aboveground 0.008; 5.020; 0.856 0.157; 1.324; 2.252 0.048; 3.791; 0.380 0.962; <0.001; 0.706 Note: Bold numbers are signiﬁcant (P < 0.05). Table 1. Infauna We detected signiﬁcant effects of experimental manipulations on infauna after 90 and 270 d (Tables 1 and 2; Appendix S2: Tables S1, S2). After 90 d, total abundance of infauna increased in the aboveground (dowel) plots in the mudﬂat March 2017 ❖Volume 8(3) ❖Article e01711 ❖www.esajournals.org 7 REYNOLDS ET AL. Table 2. A priori planned contrasts of structural manipulations on sediment characteristics across habitats for 90 and 270 d after experimental initiation, and 30 d after experimental removal. Response Estimate SE t-Value Pr(>\|t\|) Benthic microalgae Habitat (mudﬂat vs. hybrid) 0.813; 0.395; 0.366 0.342; 0.250; 0.148 2.377; 1.587; 2.468 0.023; 0.121; 0.019 Habitat ctrl vs. dowel 0.866; 0.234; 0.570 0.590; 0.399; 0.256 1.468; 0.711; 2.226 0.151; 0.482; 0.033 Hybrid ctrl vs. aboveground 0.523; 0.154; 0.295 0.475; 0.413; 0.257 1.100; 0.374; 1.148 0.279; 0.711; 0.259 Hybrid ctrl vs. belowground 0.494; 0.274; 0.364 0.408; 0.309; 0.199 1.211; 0.886; 1.830 0.234; 0.381; 0.076 Mudﬂat ctrl vs. aboveground 0.658; 0.270; 0.818 0.510; 0.370; 0.239 1.290; 0.728; 3.415 0.206; 0.471; 0.002 Sediment organic matter Habitat (mudﬂat vs. hybrid) 1.172; 0.691; 0.677 0.263; 0.143; 0.148 4.452; 4.846; 4.579 <0.001; <0.001; 0.002 Habitat ctrl vs. dowel 0.170; 0.170; 0.205 0.382; 0.210; 0.231 0.445; 0.807; 0.887 0.659; 0.425; 0.381 Hybrid ctrl vs. aboveground 0.519; 0.078; 0.033 0.333; 0.210; 0.222 1.556; 0.372; 0.151 0.128; 0.712; 0.881 Mudﬂat ctrl vs. aboveground 0.078; 0.073; 0.163 0.329; 0.170; 0.189 0.238; 0.431; 0.866 0.813; 0.669; 0.392 Hybrid ctrl vs. belowground 1.048; 0.217; 0.417 0.323; 0.194; 0.206 3.240; 1.121; 0.023 0.002; 0.270; 0.051 Silt-to-sand ratio Habitat (mudﬂat vs. hybrid) 1.999; 1.742; 1.845 0.387; 0.300; 0.556 5.168; 5.800; 3.319 <0.001; <0.001; 0.002 Habitat ctrl vs. dowel 0.658; 0.183; 0.569 0.514; 0.446; 0.732 1.279; 0.411; 0.777 0.208; 0.684; 0.442 Hybrid ctrl vs. aboveground 0.308; 0.020; 0.542 0.323; 0.301; 0.565 0.951; 0.066; 0.960 0.347; 0.948; 0.343 Mudﬂat ctrl vs. aboveground 0.401; 0.078; 0.800 0.482; 0.410; 0.648 0.831; 0.190; 1.234 0.411; 0.850; 0.225 Hybrid ctrl vs. belowground 0.539; 0.797; 0.905 0.313; 0.290; 0.522 1.722; 2.749; 1.735 0.093; 0.009; 0.091 Note: Bold numbers are signiﬁcant (P < 0.05). The composition of the infaunal community in terms of trophic feeding mode (surface vs. sub- surface feeding) was inﬂuenced by habitat type (Appendix S2: Tables S5–S7). After 270 d, we found a signiﬁcant effect of dowels on surface feeders, but not on subsurface feeders, in both the mudﬂat and hybrid zones (Fig. 2C, D; Appendix S2: Table S6). Infauna However, we found no signiﬁcant effect on subsurface feeders in either zone. When quantifying the percent change in the abundance of surface and subsurface feeders (calculated as the difference in the response to dowel treatments in mudﬂat vs. hybrid zones), we found a greater increase in subsurface feeders in the hybrid zone relative to almost no increase in subsurface feeders in the mudﬂat zone (Appendix S3: Table S1, Fig. S1). The relative inﬂuences of speciﬁc taxa on these differences among aboveground vs. control plots (D vs. UC) in the mudﬂat and in the hybrid zones can be seen in the NMDS plot (Appendix S3: Fig. S2). structure (dowels) promoted total infauna in the mudﬂat. Aboveground structure also promoted annelids in the hybrid zone (Fig. 1F) and was likely driven by changes in polychaete, but not oligochaete, taxa (Table 1). Total abundance of small crustaceans (amphipods, tanaids, excluding crabs) did not differ between the two habitats, but did increase in the presence of aboveground structure across and within habitats (Fig. 1G). While there were no observable effects of habitat or treatment on infaunal species richness after 90 d, at 270 d species richness was higher in the mudﬂat and increased with aboveground and belowground manipulations in hybrid zone compared to unmanipulated plots (Fig. 1H; Appendix S2: Table S4). Some treatment effects persisted 30 d after the removal of the structural manipulations (Table 1; Appendix S2: Table S3). Total infaunal abun- dance remained higher in the plots that had experienced aboveground structural manipula- tions (dowel, shade) in the hybrid zone (Fig. 1I). While no differences in total annelid abundance were detected (Fig. 1J), non-decapod crustaceans remained higher in hybrid plots where above- ground structure had been previously manipu- lated (Fig. 1K). Prior structural treatments did not inﬂuence infaunal species richness according to a priori predictions (Fig. 1L, Table 1). Epifauna Epifauna densities, primarily the invasive East- ern mudsnail (Ilyanassa obsoleta), were high after 270 d within the dowel addition treatment in both habitats but were very low in the UC plots (Fig. 3). There was a similar, but much weaker, trend for native shore crabs Hemigrapsus oregonensis within March 2017 ❖Volume 8(3) ❖Article e01711 ❖www.esajournals.org March 2017 ❖Volume 8(3) ❖Article e01711 8 REYNOLDS ET AL. the dowel addition plots (Fig. 3). Other epifauna l d U l i i C i d All epifauna began disappearing from the b d l h 48 h f Fig. 1. Generally, after 90 d of treatment application, neither habitat nor aboveground Spartina-mimicking structures inﬂuenced infaunal species abundance or species richness per core (A–D). After 270 d, habitat type (mudﬂat, eradicated hybrid zone) and/or structural manipulations inﬂuenced the abundance of some infauna (E–H). These responses generally disappeared within 30 d of manipulation removal (I–L), while habitat effects generally persisted. UC, unmanipulated control plots; and D, dowel; S, shade; L, litter addition. Fig. 1. Generally, after 90 d of treatment application, neither habitat nor aboveground Spartina-mimicking structures inﬂuenced infaunal species abundance or species richness per core (A–D). After 270 d, habitat type (mudﬂat, eradicated hybrid zone) and/or structural manipulations inﬂuenced the abundance of some infauna (E–H). These responses generally disappeared within 30 d of manipulation removal (I–L), while habitat effects generally persisted. UC, unmanipulated control plots; and D, dowel; S, shade; L, litter addition. the dowel addition plots (Fig. 3). Other epifauna including Urosalpinx cinerea, Carcinus maenas, and Haminoea japonica were present but not abundant enough for analysis. All epifauna began disappearing from the aboveground structure plots within 48 h of dowel removal (direct observation); after 30 d, only two Ilyanassa were found across all plots the dowel addition plots (Fig. 3). Other epifauna including Urosalpinx cinerea, Carcinus maenas, and Haminoea japonica were present but not abundant enough for analysis. All epifauna began disappearing from the aboveground structure plots within 48 h of dowel removal (direct observation); after 30 d, only two Ilyanassa were found across all plots ❖www.esajournals.org March 2017 ❖Volume 8(3) ❖Article e01711 9 REYNOLDS ET AL. Fig. 2. Response of taxa by trophic mode per core after 90 (A, B) and 270 (C, D) days of treatment application. No treatment or habitat effects persisted 30 d after experimental removal (E, F). Epifauna UC, unmanipulated control plots; and D, dowel; S, shade; L, litter addition. Fig. 2. Response of taxa by trophic mode per core after 90 (A, B) and 270 (C, D) days of treatment application. No treatment or habitat effects persisted 30 d after experimental removal (E, F). UC, unmanipulated control plots; and D, dowel; S, shade; L, litter addition. and habitats (Fig. 3F). These species readily dis- perse across mudﬂats and are present only at low density except within structured habitat (Appendix S4: Table S1). For the predation assays, after 24 h, almost all tethered Ilyanassa were recovered alive (372 out of 378 total snails) regardless of whether they were inside dowel or UC plots across the mudﬂat and hybrid zone. However, very few non-tethered (freely moving) snails were recovered from any plots (four out of 400 individuals), indicating high dispersal poten- tial for these adult snails. loss of 70.4 30 g per plot). Structural manipula- tion, however, had no effect on the ﬁnal abun- dance of decaying root mat material (F4 = 0.270, P = 0.896), which differed across blocks (F1 = 9.000, P = 0.005). We ﬁnd that this rate of decline in belowground biomass is similar to the rate of breakdown measured in long-term plots at three other sites, which shows that belowground biomass may take 6 yr or more to completely break down (Appendix S5: Fig. S1). Benthic microalgae Benthic chlorophyll-a was not affected by treat- ment, but did vary across blocks and was greater in the hybrid compared to the mudﬂat zone at 90 d (Table 2, Fig. 4A). Thirty days following structure removal, benthic microalgae was again inﬂuenced by spatial block and was greater in the Sediment organic content After 90 d, sediment organic matter was greater in the hybrid zone compared to the mudﬂat (Table 2, Fig. 4B). This pattern persisted after 270 d, but did not vary among structural manipu- lations (Table 2, Fig. 4D). This result was also observed at 90 and 270 d for sediment grain size fractionation (greater silt accumulation in the hybrid). Thirty days after the structure removals, sediment organic content remained higher in the hybrid compared to the mudﬂat habitat (Table 2, Fig. 4F); this result was consistent with the greater fraction of silt found in the sediment in the hybrid. Belowground biomass Belowground biomass of the hybrid Spartina root mat in UC plots in the eradicated hybrid zone declined on average 51% from the start until the end (day 270) of the experiment (P < 0.001, March 2017 ❖Volume 8(3) ❖Article e01711 ❖www.esajournals.org 10 REYNOLDS ET AL. Fig. 3. The two most abundant epifauna species, the crab Hemigrapsus oregonensis per pit fall trap (A, C, E) and the snail Ilyanassa obsoleta per m2 in surveys (B, D, F) within experimental plots at the middle (day 90) and end (day 270) of the experiment, and 30 d after the aboveground structure (dowels), were removed. Ilyanassa were rare to absent at day 0 and after 30 d of recovery. The day 90 and the recovery samplings occurred in early fall and spring, respectively, when the crabs are active. The day 270 sampling occurred in the winter when crabs are burrowed and less active. UC, unmanipulated control plots; and D, dowel plots. Fig. 3. The two most abundant epifauna species, the crab Hemigrapsus oregonensis per pit fall trap (A, C, E) and the snail Ilyanassa obsoleta per m2 in surveys (B, D, F) within experimental plots at the middle (day 90) and end (day 270) of the experiment, and 30 d after the aboveground structure (dowels), were removed. Ilyanassa were rare to absent at day 0 and after 30 d of recovery. The day 90 and the recovery samplings occurred in early fall and spring, respectively, when the crabs are active. The day 270 sampling occurred in the winter when crabs are burrowed and less active. UC, unmanipulated control plots; and D, dowel plots. Porewater sulfide hybrid compared to the mudﬂat and was lower in plots previously exposed to belowground mani- pulation (Table 2, Fig. 4C). Porewater sulﬁde levels were signiﬁcantly affected by habitat type. Sulﬁdes were higher in the hybrid zone (6.561 ppm 5.344 SD) relative to the mudﬂat (1.645 ppm 1.359 SD; t test: t = 2.424, df = 12.933, P < 0.031). DISCUSSION Our results show that the ghost of invasion past is present in this system through persistent legacy effects that can strongly inﬂuence habitat recovery and the potential for future restoration. By comparing eradicated hybrid Spartina habi- tats to nearby uninvaded (i.e., pre-invasion con- dition) mudﬂat, we found that the legacy effects of invasive ecosystem engineers can persist for March 2017 ❖Volume 8(3) ❖Article e01711 ❖www.esajournals.org March 2017 ❖Volume 8(3) ❖Article e01711 11 REYNOLDS ET AL. Fig. 4. Local heterogeneity (i.e., block effects) had a larger effect on benthic microalgae (A, C, E) across time points (see Appendix S2: Tables S1–S3). Habitat type and/or structural manipulations inﬂuenced sediment organic matter (ash-free dry mass per core; B, D, F) after 270 d and 30 d after manipulation removal. The pattern for the ratio of sediment silt to sand (not pictured) followed the same patterns as sediment organic matter. UC, unmanipulated control plots; and D, dowel; S, shade; L, litter addition. Fig. 4. Local heterogeneity (i.e., block effects) had a larger effect on benthic microalgae (A, C, E) across time points (see Appendix S2: Tables S1–S3). Habitat type and/or structural manipulations inﬂuenced sediment organic matter (ash-free dry mass per core; B, D, F) after 270 d and 30 d after manipulation removal. The pattern for the ratio of sediment silt to sand (not pictured) followed the same patterns as sediment organic matter. UC, unmanipulated control plots; and D, dowel; S, shade; L, litter addition. been eradicated), we found that a much broader range of species, including annelids (mostly polychaetes) as well as crustaceans (mostly amphipods), increased in abundance in response to experimental aboveground structure addi- tions. By contrast, only crustaceans (mostly amphipods) showed a signiﬁcant increase in abundance in response to the dowel manipula- tion in the mudﬂat zone. These results agree with our predictions that subsurface feeders, like many annelids, would recruit to dowel treat- ments if the belowground conditions were still similar to the invaded conditions as the result of legacy effects. By contrast, we saw no increase in the subsurface feeders in the mudﬂat despite adding dowels, likely because the belowground conditions were not conducive for these species. ❖www.esajournals.org March 2017 ❖Volume 8(3) ❖Article e01711 DISCUSSION In their native range, the mussel can live for more than a decade (Bertness and Grosholz 1985), the whelk can live 6–10 yr (Franz 1971), the green crab can live up to 6 yr, and the mudsnail can live for as long as 70 yr (Curtis et al. 2000). Given that there is rela- tively little predation pressure on invasive mussels and snails in this system (Fig. 3; Weiskel 2012), adult survival may be high. While the persistent belowground structure still maintains populations of the mussel, the absence of aboveground struc- ture may over time have negative effects on epifaunal reproduction and survivorship by removing important egg-laying habitat for snails and sediment stabilization for the mussel. Despite the now complete absence of the aboveground effects of hybrid Spartina at this site, even if repro- duction was substantially reduced (or even failed completely), long-lived adult populations of invasive epifauna could persist for many years or, in the case of the invasive mudsnail I. obsoleta, for many decades beyond the aboveground engineering effects of the hybrid Spartina. We found support for this contention due to the rapid recruitment of adult mudsnails into our experimental plots with the addition of the struc- tural manipulations, despite the time since the eradication of their hybrid Spartina habitat. There- fore, measuring eradication success may require intensive monitoring combined with targeted experimental approaches to capture changes occurring at slower-than-expected rates. p f Our experimental results point to two mecha- nisms as most important in contributing to the observed delays in recovery of the invaded sys- tem to pre-invaded conditions following eradica- tion of hybrid Spartina. The ﬁrst is the effect of the belowground structure consisting of an extensive network of roots and rhizomes that can remain for several years as they undergo slow breakdown in the highly reduced sediments typ- ical of these habitats. This can produce persistent effects where the decaying roots and rhizomes provide trophic support and belowground habi- tat for small detritivores, but also persistent ele- vated levels of porewater sulﬁde, which can also negatively affect infaunal communities (Neira et al. 2006). We found that some sediment char- acteristics were slow to change, such as sedi- ment organic matter and the silt-to-sand ratio. Additionally, despite several years post-hybrid Spartina eradication signiﬁcant differences per- sisted across control plots in the two habitats. DISCUSSION The recruitment of crustaceans to both habitats supports the idea that highly mobile epibenthic species like amphipods, which used refugia several years post-eradication as a function of both belowground impacts that are slowly disap- pearing, but still present, and aboveground effects that disappeared quickly but whose impact continues in the form of surviving long- lived epifaunal invader populations. We suggest that both of these mechanisms are likely to be general in systems involving invasive plants as ecosystem engineers. Our work is among the ﬁrst to quantify the change, or lack of change, in food web and community processes in an estuarine/ marine system following the removal of an ecosystem engineer, as well as to quantify several of the mechanisms that contribute to persistent engineering effects after invader eradication. Our manipulative ﬁeld experiments showed distinct differences between habitats as well as differences among taxa in response to experi- mental additions of aboveground structural mimics. In the hybrid zone (where Spartina had ❖www.esajournals.org March 2017 ❖Volume 8(3) ❖Article e01711 12 REYNOLDS ET AL. provided by Spartina stems during the invasion (Neira et al. 2006), would now rapidly recruit to experimentally re-created aboveground structure if still in the system. We found that many of these species are still present in large numbers, although now, in the absence of the aboveground refugia, are widely dispersed at low density. Overall, these results support the idea that the effects of the roots and rhizomes are undergoing slow decay, and that their persistent effects on sediment characteristics and porewater chem- istry are maintaining a legacy effect of the inva- sion in the hybrid zone strongly affecting the benthic community and food web. This raises the possibility of rapid re-assembly of the invaded community if this habitat is reinvaded by hybrid Spartina or another comparable plant in the near future, which may have consequences for native Spartina foliosa restoration activities. The second mechanism concerns the presence of long-lived members of the invaded community that were facilitated by the effects of the above- ground structure (Neira et al. 2006, Grosholz et al. 2009). Species facilitated by the hybrid Spartina invasion included long-lived non-native species, such as the eastern ribbed mussel (Geukensia demissa), the eastern mudsnail (Ilyanassa obsoleta), the eastern whelk Urosalpinx cinerea, and the Euro- pean green crab (Carcinus maenus). March 2017 ❖Volume 8(3) ❖Article e01711 ❖www.esajournals.org DISCUSSION Thus, the slow breakdown of this root/rhizome mat, as also observed at other locations through- out San Francisco Bay (Appendix S5: Fig. S1), may dictate the equally slow transition of the benthic food web from dominance of subsurface detritivores to the pre-invasion community dom- inated by surface-feeding consumers. This mech- anism may be most likely in hypoxic or anoxic soils or sediments where anaerobic processes dominate and breakdown of roots and rhizomes is slow. Previous studies of the consequences of remov- ing invasive ecosystem engineers typically involve terrestrial plant species and their impact on native plants (reviewed in Corbin and D’Antonio 2012). For example, hysteresis in the recovery of a variety of different ecological functions, such as the reestablishment of ecological interaction networks of native birds, was found following removal of invasive honeysuckle (Rodewald et al. 2010). However, in coastal marine and estuarine systems, March 2017 ❖Volume 8(3) ❖Article e01711 ❖www.esajournals.org 13 REYNOLDS ET AL. REYNOLDS ET AL. there are few examples of the complete eradication of invasive ecosystem engineers and even fewer examples of systems that have experimentally tested the consequences of removal on entire com- munities (McGrorty and Goss-Custard 1987, Hacker and Dethier 2009). Most research examin- ing restoration and recovery of marine or estuarine systems has focused on removal of other factors, such as ﬁshing (Blackwood et al. 2012, Collie et al. 2013) or contaminants (Valega et al. 2008), likely due to the logistical challenges of complete inva- der removal and the difﬁculty in evaluating inva- der impacts. In general, research conducted on invasive species removal in these habitats, whether or not they were engineers, supports the idea that these systems may be unable to return to the pre-invaded condition. For instance, the removal of Caulerpa racemosa in the Mediterranean Sea resulted in the failure of the original macroalgal community to reestablish (Piazzi and Ceccherelli 2006). As in most other cases, the mechanisms underlying this failure to regain the pre-invasion community remain untested. Other studies have examined recovery of native plants after removal of another invasive Spartina species (Spartina anglica), and, while the speciﬁc mechanisms remain undocumented (Hacker and Dethier 2009), we assume that they are similar to our ﬁndings. of aboveground structure may be the persistence of secondary invaders and other species for which that structure played a facultative role. DISCUSSION Despite the relatively immediate loss of that habitat, long-lived species (such as the invasive mudsnails) may continue to persist, often in more resistant adult stages, even if conditions are not conducive to reproduction in the absence of the habitat created by the engineer. While sur- veys may reveal low levels of the secondary invaders and the restoration thereby determined a success, this designation may be premature as the secondary invaders may be retained in the system and their populations grow rapidly with the reestablishment of the invasive ecosystem engineer or a functionally similar native or exotic vegetation. g Finally, we argue that the delayed transition to the pre-invasion state we have documented may complicate evaluations of restoration success. Most of the current theory regarding the failure to reestablish the pre-invasion community, whether native or a mix of native and non-native, has focused on the presence of alternate states (or even alternate stable states) and the problems this may pose for evaluating restoration success (e.g., Suding et al. 2004). The slow transition to the pre-invasion state may suggest the presence of alternate states, especially when the eradicated invader was an ecosystem engineer with strong legacy effects. If a system, such as that examined here, required many years to recover the pre- invasion condition and was monitored with inadequate frequency or duration, it could be mistaken for one that is in a persistent alternate state rather than one moving slowly toward recovery of the pre-invasion state. The distinct roles of both above- and below- ground plant structure that we have demon- strated are likely to generalize to other similar systems involving invasive plants. In other sys- tems such as bogs, swamps, or boreal habitats where breakdown of belowground plant bio- mass (roots, rhizomes, etc.) is delayed, this could also lead to persistent effects of ecosystem engineers well beyond the date of eradication. Additional impacts on nutrient cycling and enhancement of porewater sulﬁde could further add to delays in recovery. As we documented in our experimental manipulations, these delays could inﬂuence soil fauna and characteristics along with the recovery of native vegetation in systems with prolonged belowground break- down. The effects of aboveground structure are likely to be more shortlived with respect to the physical characteristics, although it may have the potential for long-term effects dependent on the rate of breakdown of leaves, shoots, branches, etc., in the surface litter. ❖www.esajournals.org March 2017 ❖Volume 8(3) ❖Article e01711 DISCUSSION We suggest that one of the most important long-term impacts In summary, we conclude that the ghost of invasion past is operating in this system through persistent legacy effects of both above- and belowground effects that not only inﬂuence the state of recovery but also the potential for future restoration. These legacy effects arise from belowground processes that include the slow breakdown of roots and rhizomes creating belowground detritus and high levels of porewa- ter sulﬁde that support the persistence of the invaded belowground community. Legacy effects also arise from long-lived epifaunal species that were facilitated by the refugia created by the aboveground structure and that now can persist March 2017 ❖Volume 8(3) ❖Article e01711 March 2017 ❖Volume 8(3) ❖Article e01711 ❖www.esajournals.org 14 REYNOLDS ET AL. for biodiversity. Trends in Ecology and Evolution 17:40–44. for many years, if not decades, despite the disap- pearance of this refugia. We suggest that both of these mechanisms are likely to be general in many similar systems involving invasive plants that are ecosystem engineers. This includes many terrestrial systems and aquatic habitats with soils that are poorly aerated and have limited capaci- ties for breakdown of belowground plant mate- rial. Also, invasive plants in many other systems may also attract longer-lived non-native species, including beetles, ﬁshes, reptiles, and rodents. Our work also points to the likelihood that many systems may respond much more slowly to the removal of invasive ecosystem engineers than expected, and may follow a slower-than-expected trajectory toward recovery of the pre-invasion food web. This slow transition would be missed if the monitoring interval or duration is insufﬁ- cient to capture the rate of change. We suggest that short-term manipulation of either above- or belowground engineering structures are an efﬁ- cient means of determining how far a system is from both the invaded and pre-invasion commu- nity, and may provide insights into the time required for recovery and restoration success. Collie, J. S., W. L. Adamowicz, M. W. Beck, B. Craig, T. E. Essington, D. Fluharty, J. Rice, and J. N. Sanchirico. 2013. Marine spatial planning in prac- tice. Estuarine, Coastal and Shelf Science 117: 1–11. Corbin, J. D., and C. M. D’Antonio. 2012. Gone but not forgotten?: invasive plants’ legacies on community and ecosystem properties. Invasive Plant Science and Management 5:117–124. Crooks, J. 2002. Characterising ecosystem-level conse- quences of biological invasions: the role of ecosys- tem engineers. Oikos 97:153–166. ACKNOWLEDGMENTS We would like to thank Charles Norton, Andre San- chez, Katrina Sutton, Sara Hutton, Erica Pollard, Samantha Linhardt, Rachel Wigginton, and Jason Sad- owski for ﬁeld and laboratory assistance. We would like to thank the National Science Foundation CNH Program (DEB-1009957) for funding. EG, PLR, and SY designed the experiments. PLR, EG, JG, and CH per- formed the ﬁeldwork, and PLR, JG, CH, and EG pro- cessed all samples. JC contributed logistical support. PLR and EG analyzed the data and wrote the manu- script. We thank two anonymous reviewers for their insights, which improved this manuscript. Grosholz, E. 2005. Recent biological invasion may has- ten invasional meltdown by accelerating historical introductions. Proceedings of the National Acad- emy of Sciences USA 102:1088–1091. Grosholz, E., L. Levin, A. Tyler, and C. Neira. 2009. Changes in community structure and ecosystem function following Spartina alterniﬂora invasion of Paciﬁc estuaries. Pages 23–40 in B. Silliman, E. Grosholz, and M. Bertness, editors. Human impacts on salt marshes. University of California Press, Berkeley, California, USA. Grove, S., K. Haubensak, and I. Parker. 2012. Direct and indirect effects of allelopathy in the soil legacy of an exotic plant invasion. Plant Ecology 213: 1869–1882. DISCUSSION Curtis, L., J. Kinley, and N. Tanner. 2000. Longevity of oversized individuals: growth, parasitism, and history in an estuarine snail population. Journal of the Marine Biological Association of the UK 80: 811–820. D’Antonio, C., F. Hughes, M. Mack, D. Hitchcock, and P. Vitousek. 1998. The response of native species to removal of invasive exotic grasses in a seasonally dry Hawaiian woodland. Journal of Vegetation Science 9:699–712. Franz, D. R. 1971. Population age structure, growth and longevity of the marine gastropod Urosalpinx cinerea. Biological Bulletin 140:63–72. g Gallardo, B., M. Clavero, M. Sanchez, and M. Vila. 2016. Global ecological impacts of invasive species in aquatic ecosystems. Global Change Biology 22:151–163. LITERATURE CITED Bertness, M., and E. Grosholz. 1985. Population dynamics of the ribbed mussel, Geukensia demissa: the costs and beneﬁts of an aggregated distribu- tion. Oecologia 67:192–204. Grove, S., I. Parker, and K. Haubensak. 2015. Persistence of a soil legacy following removal of a nitrogen-ﬁxing invader. Biological Invasions 17: 2621–2631. Hacker, S., and M. Dethier. 2009. Differing conse- quences of removing ecosystem-modifying inva- ders: signiﬁcance of impact and community context to restoration potential. Pages 375–385 in G. Rilov and J. Crooks, editors. Biological invasions Blackwood, J., A. Hastings, and P. Mumby. 2012. The effect of ﬁshing on hysteresis in Caribbean coral reefs. Theoretical Ecology 5:105–114. Coleman, F., and S. Williams. 2002. Overexploiting mar- ine ecosystem engineers: potential consequences March 2017 ❖Volume 8(3) ❖Article e01711 ❖www.esajournals.org 15 REYNOLDS ET AL. REYNOLDS ET AL. in marine ecosystems. Springer-Verlag, Berlin, Heidelberg, Germany. assemblages after removal of Caulerpa racemosa var. cylindracea. Estuarine, Coastal and Shelf Science 68: 655–661. Holsman, K., P. McDonald, P. Barreyro, and D. Arm- strong. 2010. Restoration through eradication: Removal of an invasive bioengineering macro- phyte (Spartina alterniﬂora) restores some habitat function for a native mobile predator (Cancer mag- ister). Ecological Applications 20:2249–2262. R C R Core Team. 2015. R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project. org/ Reeder, T. G., and S. Hacker. 2004. Factors contributing to the removal of a marine grass invader (Spartina anglica) and subsequent potential for habitat restoration. Estuaries 27:244–252. g pp Kwak, T. J., and J. B. Zedler. 1997. Food web analysis of southern California coastal wetlands using multiple stable isotopes. Oecologia 110:262–277. p p g Levin, L., C. Neira, and E. Grosholz. 2006. Invasive cordgrass modiﬁes wetland trophic function. Ecology 87:419–432. Ridenour, W., and R. Callaway. 2001. The relative importance of allelopathy in interference: the effects of an invasive weed on a native bunchgrass. Oecologia 126:444–450. gy Marchante, H., H. Freitas, and J. H. Hoffman. 2011. Post-clearing recovery of coastal dunes invaded by Acacia longifolia: Is duration of invasion relevant for management success? Journal of Applied Ecology 48:1295–1304. g Rodewald, A. D., D. P. Shustack, and L. E. Hitchcock. 2010. Exotic shrubs as ephemeral ecological traps for nesting birds. Biological Invasions 12:33–39. Rosso, P., S. Ustin, and A. Hastings. 2006. Use of LIDAR to study changes associated with Spartina invasion in San Francisco Bay marshes. Remote Sensing of Environment 100:295–306. Maron, J. L., and R. L. Jefferies. 2001. LITERATURE CITED Restoring enriched grasslands: effects of mowing on species richness, productivity and nitrogen retention. Ecological Applications 11:1088–1100. Sheehan, M., and J. Ellison. 2015. Tidal marsh erosion and accretion trends following invasive species removal, Tamar Estuary, Tasmania. Estuarine, Coastal and Shelf Science 164:46–55. McGrorty, S., and J. Goss-Custard. 1987. A review of the rehabilitation of areas cleared of Spartina. Insti- tute of Terrestrial Ecology (Natural Environment Research Council), Dorset, UK. Simberloff, D., and B. Von Holle. 1999. Positive interac- tions of nonindigenous species: Invasional melt- down? Biological Invasions 1:21–32. Mendez, M., E. Schwindt, and A. Bortolus. 2015. Dif- ferential benthic community response to increased habitat complexity mediated by an invasive barna- cle. Aquatic Ecology 49:441–452. g Simberloff, D., et al. 2013. Impacts of biological inva- sions: What’s what and the way forward. Trends in Ecology and Evolution 28:58–66. q gy Neira, C., E. Grosholz, L. Levin, and R. Blake. 2006. Mechanisms generating modiﬁcation of benthos following tidal ﬂat invasion by a Spartina hybrid. Ecological Applications 16:1391–1404. gy Suding, K., K. Gross, and G. Houseman. 2004. Alterna- tive states and positive feedbacks in restoration ecology. Trends in Ecology and Evolution 19: 46–53. Neira, C., L. Levin, and E. Grosholz. 2005. Benthic macrofaunal communities of three sites in San Francisco Bay invaded by hybrid Spartina, with comparison to uninvaded habitats. Marine Ecology Progress Series 292:111–126. Tyler, A., J. Lambrinos, and E. Grosholz. 2007. 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Journal of Field Ornithol- ogy 78:395–400. Weiskel, H. S. 2012. The interaction of nutrient loading and biological invasions in a benthic estuarine community. REYNOLDS ET AL. REYNOLDS ET AL. Wright, J., and C. Jones. 2006. The concept of organ- isms as ecosystem engineers ten years on: pro- gress, limitations, and challenges. BioScience 56: 203–209. Zavaleta, E., R. Hobbs, and H. Mooney. 2001. Viewing invasive species removal in a whole-ecosystem con- text. Trends in Ecology and Evolution 16:454–459. gy Zhang, M., S. L. Ustin, E. Rejmankova, and E. W. Sanderson. 1997. Monitoring Paciﬁc coast salt marshes using remote sensing. Ecological Applica- tions 7:1039–1053. Yelenik, S., and J. Levine. 2011. The role of plant-soil feedbacks in driving native-species recovery. Ecology 92:66–74. LITERATURE CITED University of California, Davis, Cali- fornia, USA. Piazzi, L., and G. Ceccherelli. 2006. Persistence of biological invasion effects: recovery of macroalgal March 2017 ❖Volume 8(3) ❖Article e01711 ❖www.esajournals.org 16 March 2017 ❖Volume 8(3) ❖Article e01711 SUPPORTING INFORMATION Additional Supporting Information may be found online at: http://onlinelibrary.wiley.com/doi/10.1002/ecs2. 1711/full nal Supporting Information may be found online at: http://onlinelibrary.wiley.com/doi/10.1002/ecs2 ❖www.esajournals.org March 2017 ❖Volume 8(3) ❖Article e01711 17
https://openalex.org/W4298437458	https://zenodo.org/records/6565937/files/453-458.pdf	English	null	MUTUAL COAGULATION OF COLLOIDAL SOLUTIONS. INTERACTION BETWEEN COLLOIDAL ANTIMONY SULPHIDE AND COLLOIDAL FERRIC HYDROXIDE AND THE EQUILIBRIUM DIALYSATE OF COLLOIDAL FERRIC HYDROXIDE	Zenodo (CERN European Organization for Nuclear Research)	1,951	cc-by	3,245	MUTUAL COAGULATION OF COLLOIDAL SOLUTIONS~. IN'fERACTION BETWEEN COLLOIDAL ANTIMONY SULPHIDE AND COLLOIDAL FERRIC HYDROXIDE AND THE EQUILIBRIUM DIALYSATE OF COLLOIDAL FERRIC HYDROXIDE BY P. M. BARVI!; .\NO A. K. ]AlloiBOTKAR By a modified process of dialysis, colloidal ferric hydroxide and its "equilibrium dialysate" of nearly the same ionic envirotunents have been compared for their powers' as coagulating agents. lt is found that in mutual coagulation the colloidal ferric bydmxide particles play a very impot'tant part, and in fact, they mAy be taken to nct·11s polyvalent cations. Dialysis of a colloidal solntion of ferric hydroxide has been a subject of numerous investigations. Ferric hydroxide sol, when prepared by the hydrolysis of ferric chloride at an elevated temperature, contains ferric chloride, which has remained on- hydrolysed, and hydrochloric add formed as a result of hydrolysis. Changes in the cornl'osilion of the intermiccllary liquid are brought about when the colloidal solution is submitted to dialysis. These changes create a marked effect on the properties of the colloidal solution as a whoie. Desai and Barve (Tt"ans. Nat. Inst. Sci. India, 1939, 2 39) have fully dealt with the ~hanges brought about by dialysis in the case o[ ferric hydroxide. They have shown therein that the cataphoretic speed ·of the colloidal particles increases, reaches a maximum, and then decreases; the conductivity, however, decreases continually, and the stability, as dt:termined by :B.occulation values with electrolytes, also decreases continually with progress of dialysis. Desai and Bcrkar (TTans. FaTaday Soc., 1933, 29, 1269), Dhar and collaborators (]. Phys. Chem., 1920, 26, 7oo) and :Mukherjee and co-workers (this Journal, I9JI, 8, 373i 1933, 10, 27) have supported the conclusions reached by Desai and Barve (loc. cit.). Among the investigations of the properties of colloidal ferric hydroxide with {lrogressive dialy!;is, the study of its power to coagulate mutually another colloid, namely antimony sulphide, was specially undertaken. The properties of antimony sulphide remained unchanged as it was not submitted to dialysis or any other treatment. It was prepared by dropping slowly a o.s% solution of tartar emetic in water, saturated with hydrogen s'.llphide through which H 2S gas was slowly bubbled, tbe excess of the gas being driven out by passing hydrogen through the sol so formed. In case of colloidal ferric hydroxide, the coagulating power is made up of the effect of the pure colloid and the electrolyt~s present in the intermicellary liquid. [jour. Tndian Chew. So:.-., \·ol. 28, No.8, r95I] [jour. Tndian Chew. So:.-., \·ol. 28, No.8, r95I] MUTUAL COAGULATION OF COLLOIDAL SOLUTIONS~. IN'fERACTION BETWEEN COLLOIDAL ANTIMONY SULPHIDE AND COLLOIDAL FERRIC HYDROXIDE AND THE EQUILIBRIUM DIALYSATE OF COLLOIDAL FERRIC HYDROXIDE The process of dialysis ''purifies" the colloid, and hence iu the later period of dialysis, the coagulating power is DJ(.Jre and more due to the colloid and less and less due to the intermicellary liquid. Exl'BkiMBNTAL To aScertain the coagulating effect of the pure colloid alone, tht: following modified process of dialysis was adopted. 454 P. ~r. DARVE AND A. K. J"f\MDOTKAR A colloidal ferric hydroxide solution was placed in a parchment paper bag and suspeti.· ded in conductivity water. The water outside the bag, in wh{cb the electrolytes of the intermicellary liquid diffused, became the dialysate and went on increasing m its conduc- tivity till1t reached. a maximum value at which it remamed stationary. This means that an equilibrium has reached bet\'l'een the colloid and the dialysate as regards the diffusion of tl1e electrolytes in the intermicellary liquid. Usually this condition was attained within 48 to 72 hours. At this stage the dialysate was removed, as also a pat·t of the colloid for quantitative analysis. 'fhe dialysate was then entirely replaced by conductivity water and the process repeated as indicated above for every stage of dialysis. This dialysate is termed the "equilibrium dialysate". q y In the p;ocess of dialysis, the dialysing membrane allows a free passage to the electrolytes present in the intermicellary liquid, bnt not to the colloid tJ&rticles. By adopting the process of dialysis, as stated above, the" distribution of the electrolytes between the colloid and the dialysate at eqt1ilibri\lm is governed by the Donnan equili- brium. On analysis of the colloid and the dialysate Mukherjee and co-workers (loc. cit.) have shown that the ultrafiltrate is poorer in chloride ions but richer in hydrogen ions and of a higher s,:ecific conductivity than that of the residual sol. 1'he coagulating power of the colloidal solution and of the "equilibrium dialysate" was measured by finding out the volume of the respective solutions required for the coagulation of 2 c.c. of the antimony sulphide sol (the total volume of the mixture being kept constant at Io c.c.). The fl~culation of this negatively charged antirn011y sulphide sol by the colloidal ferric hydroxide, dialysed to different extents, is a case of mutual coagulation, whereas the coagulation of the same antimony sulphide sol by the ''equilibrium dialysate" is a case of simple electrolytic coagulation brought about bJ the positive ioos ·of the electrolyte present in the "equilibrium dialysate". Mutual coagulation, as stated by Weiser and Chapman fJ. Phys. Exl'BkiMBNTAL Che"'·· 19,31, 35, 543, 1932, 36, 7141, is governed by (I) electrical neutralisation of the charge, (2} mutual adsorption of the colloidal particles, (3) interaction between stabilising ions and (4·1 presence of the excess of free electrolytes in the sol. The various results obtained by adopting the above process of dialysis gave a better insight intQ the mecbaDism of mutual coagulation. Stage of dialysis. 2 3 4 5 ,6 7 8 DAys from the start. 0 5 JO r3 J6 19 ,:ar a6 ConducCi'Vily. Sp. cnnda<"tance Colloid. F.q. dial~·sate. 37.8oxzo·9 r7.30 Jj.3?'X tr:.·i IO.OO ro.oo 7.20 7-30 4-SIS .IJ.6.; 3-33 2.9I 2-70 2.!10 2.65 2.10 455 )JU'l'U,\L l.:OAGULATION OF COLLOIDAL SOLUTIONS, ETC. Stage o{ dialysis. 1 J 3 4 g 7 8 Stage u( olialysis. .z 3 4 5 6 7 8 TABU;: II Free iron and c1zlo·ride content. llree irou content Colloid. Eq. dialysate. 4.:~o g./litre 2.24 o.84 o.36 (),164 0.0/5 0.011 1.12 g./litre 0.78 0-34 0112 o.o6o o.oo8 TAnr.r. Ill Charge em l'olloid particles 27.6 X ·&em /Volt/c11J./sec. 40-1 55·3 75-0 8:z 8 5t..S 42.1 JO.I DISCUSS19N Chloride content Colloid. Eq. dialysate. 9·55 g./litre 4 41l . 2.20 1-42 0-99 0.166 O.OJ6 o.o:zo J·S'l g./litre I.J2 O.QO 0.46 0.099 o.o:Z7 o.OIO Coagulation values Colloid. F.q. dialysate. o.:zo c.c. 0-40 0-55 0-7$ o.B,; i.15 1.25 1-45 0-55 c.c . 0 75 1.85 590 TABU;: II Free iron and c1zlo·ride content. TABU;: II 'rhe dial}· sis has been carried out in eight stages covering 26 days. The conductivity ('!'able I, Fig. r), it will be observed, decreases continually in both th.: cases 'fhc: conductivity of the colloid, bowe,.·er, does not differ much from that of the •'equilibrium dialysate". The free iron content of the colloid and that of the "equilibrium dialysate" are given in Tablt: II and Fig 2. It will be observed that the concentration of the free iron in both of them is very nearly equal, showing thereby that the ionic environment of both the colloid and the "equilibrium dialysate", as far as the ferric ions are concerned, is nearly the same. Along with the iron content, the chloride content of the colloid and the "t:qUili· brium dialysate" is given in Table II. It is clear, that though the chloride ion concen· tration in the colloid is considerably higl1er than that of ttje "equilibrium dialysate", particularly in the earlier stages of dialysis, both of them tend to zero. Exl'BkiMBNTAL This may be due to the adsorption of the chloride ions on the outer surface of the electrical double layer. 'rbe cataphoretic speed of the colloidal particles increases, ":'eacbes a maximum in the fifth st;ge (x6 days) and then decreases, with progress of dialysis. This supports the observations of Desai and Barve Cloc. cit.). '!'be values obtained for the mutual coagulation of :1 c.c. of the negatively charged antimony sulphide sol, by the colloidal ferric hydroxide and those obtained Cor the coagu. lation of au equal amount of the same antimony .sulphide sol by the "equilibrium dialysate" are ~iven in 'l'able HI. In the case of tbe coagulation of the antimony sulphide 456 5 I'. ~L R\RVt: A:XD .-\. K. J.UIDOTK~\R FIG. I. Coud~tclauu o/ colloidal F(OBl3 and rq. dialysate. COLLOID --c»---o--• 01ALY$ATIE SlageJ: of eq111ltbJ·ium. IJy the "equilibrium dialysate'', it will he oLsen•ed tl!at the coagulation values can bt obtained up to the fiith stage (16 days) of dialysis only. After this period the coagulating power of the "equilibrium dialysate' • is incapable of coagulating the sol. This clearly shows th ot the concentration of the electrolytes in the "equilibrium· dialysate" progressively decreases to such a low value that it cannot coagulate the antimony sulphide sol at all. Th l bt i d f th t l l ti f th ti l hid l b 456 5 I'. ~L R\RVt: A:XD .-\. K. J.UIDOTK~\R FIG. I. Coud~tclauu o/ colloidal F(OBl3 and rq. dialysate. COLLOID --c»---o--• 01ALY$ATIE SlageJ: of eq111ltbJ·ium. IJy the "equilibrium dialysate'', it will he oLsen•ed tl!at the coagulation values can bt obtained up to the fiith stage (16 days) of dialysis only. After this period the coagulating power of the "equilibrium dialysate' • is incapable of coagulating the sol. This clearly shows th ot the concentration of the electrolytes in the "equilibrium· dialysate" progressively decreases to such a low value that it cannot coagulate the antimony sulphide sol at all. IJy the "equilibrium dialysate'', it will he oLsen•ed tl!at the coagulation values can bt obtained up to the fiith stage (16 days) of dialysis only. After this period the coagulating power of the "equilibrium dialysate' • is incapable of coagulating the sol. This clearly shows th ot the concentration of the electrolytes in the "equilibrium· dialysate" progressively decreases to such a low value that it cannot coagulate the antimony sulphide sol at all. Exl'BkiMBNTAL The values obtained for the mutual coagulation of the antimony sulphide sol by the colloidal ferric hydroxide go on increasing gradually with its progressive dialysis and no maximum is observed. It may be obsen-cd that dunng this period of dialysis the cataphoretk speed incr,f;!ases, reaches a maximum and then decreases. In the present investigation, allowing. for a slight variation m the distribution of the electrolytes between the colloid and the "equilibrium dialysate'' due to the influence of the Donnan equilibrium, one can estimate, virtually, the effect of the intermicellary liquid alone and the effect of the same along wi~h the pure colloid partide,; that are present in the sol. By subtrac- ting one effe:ct from the other, one can get a fairly good idea of the individual coagulating effect of the pure colloidal particles. As has been mentioned in the introduction, in nmtual coagulation any one of the factors or all of them may work simultaneously. The interaction of stabilising ions does not arise here as both the colloids, ferric hyc!roxide and antimony sulphide, are acidic. The coagulation of the antimony sulphide by the "equilibrium dialysate" is 457 :1.11JTU:\t COAGULATION OF COI.T-OIDAL SOLUTIONS, ETC. :1.11JTU:\t COAGULATION OF COI.T-OIDAL SOLUTIONS, ETC. .; ~ ~ § ... s: a FIG. 2. TJtal Fe··· and Cl" in the colloid antf eq. dialysate. 4 I ' I I . \ I \ I 6 5 , \ I I ' I I I I I I ............... n' ,. Wc...m . _...,._....,._ n~ .. .,.,,.,,.,,. --1'-· ........... , •.. • C......1D • • a Fi- 1111 ..... un.e. Stages of equilfbriJim. 45 .; ~ ~ § ... s: a FIG. 2. TJtal Fe··· and Cl" in the colloid antf eq. dialysate. 4 I ' I I . \ I \ I 6 5 , \ I I ' I I I I I I ............... n' ,. Wc...m . _...,._....,._ n~ .. .,.,,.,,.,,. --1'-· ........... , •.. • C......1D • • a Fi- 1111 ..... un.e. Stages of equilfbriJim. .; ~ ~ § ... s: a 4 I ' I I . \ I \ I 6 5 , \ I I ' I I I I I I ............... n' ,. Wc...m . _...,._....,._ n~ .. .,.,,.,,.,,. --1'-· ........... , •.. • C......1D • • a Fi- 1111 ..... un.e. Stages of equilfbriJim. a case of simple coagulation by electrolytes. Exl'BkiMBNTAL As antimony sulphide sol is negatively charged, only the positively charged ions, like the ferric ions, will be effective in coagula- tion. The concentration of the ferric ions in the colloidal solution and the "equ~librium dialysate" is nearly the same. If only the ferric ions were responsible for coagulation, the values obtained with both of them ought to hav~ beo.?n almost the same. The colloidal particl~ are, however, acting as coagulating ions. Hence, in the case of the colloid, the coagulating power is the combined effect of the ferric ions and the colloidal particles. As the dialysis proceeds, the effect of the ferric ions continually diminishes. The ind1vidual effect of the ferric ions alone may be take~ to be equal to the effect of the .. equilibrium dialysate" as the concentration of the ferric ions in both of them is nearly the same. As the "equilibrium dialysate", obtained after the fifth stage of dialysis, ceases to coagulate the antimony sulphide sol, the values obtained for the colloid after this period may be taken to be the effect of the colloid particles alone. As the size of the colloid particles, as compared with the ferri~ chloride molecules, is very Stages of equilfbriJim. Stages of equilfbriJim. DEP.\RTMIN'l 01 CHEMIStRY, Wr.UOlf eor.!,.IGJI, Do!I.IBAY. Stages of equilfbriJim. after the fifth state of dialysis, it is found that the charge on the colloid decreases and the amount of the colloidal solution required for coagulating the same amount of the antimony sulphide sol i!;lcreases. This is what one would expect on theoretical grounds, as both the charge and the concentration of the ferric ions are decreasing. The constitution of the antimony sulphide sol r~mains unchanged as it is not subjected to dialysis, and hence, the effect of the antimony !"!Ulphide sol on the colloidal ierric hydroxide may be tak~p to be· constant. It should, however, be emphasized that the process of coagulation being not so simple, the difference between the two values is not necessarily an exact indication of the effcc·t of colloidal particles alone. It is qaite sufficent though to give one a clear idea about the influence of colloidal ferric hydroxide particles in mutual coagttlation. 458 P. M. UARVE AND A. 1<. J.\\lBO'fKAR FIG. 3 Coagulatiou va!l•e of tlu: colloid of dialysate aud cat. s{>cctt of colloid. liS ·= "6 ... X t:' - ~ I / r I l I I l r I r .. \ \ I I I I I I I I I 1 I -·---·-c ..... .s,. .. ....,....__,._r .... ~ ........._.._., • ·P....uf4•.1 ' \ ., .... ' ' '"', ~~ 7 8 SlaJ!I~S of equlllbrtotm. FIG. 3 Coagulatiou va!l•e of tlu: colloid of dialysate aud cat. s{>cctt of colloid. liS ·= "6 ... X t:' - ~ I / r I l I I l r I r .. \ \ I I I I I I I I I 1 I -·---·-c ..... .s,. .. ....,....__,._r .... ~ ........._.._., • ·P....uf4•.1 ' \ ., .... ' ' '"', ~~ 7 8 SlaJ!I~S f lllb t t Coagulatiou va!l•e of tlu: colloid of dialysate aud cat. s{>cctt of colloid. SlaJ!I~S of equlllbrtotm. SlaJ!I~S of equlllbrtotm. large, numerous ferric chloride molecules must ba.ve ttndergoue hydrolysis to build the colloidal 1enic hydroxide particle. The number of colloid particles so formed will naturally be extremely small in comparison with the n'umber of ferric ions which are used up. In spite of small numbe1, colloidal ferric hydroxide particles are highly effective in coagulation. Stages of equilfbriJim. a case of simple coagulation by electrolytes. As antimony sulphide sol is negatively charged, only the positively charged ions, like the ferric ions, will be effective in coagula- tion. The concentration of the ferric ions in the colloidal solution and the "equ~librium dialysate" is nearly the same. If only the ferric ions were responsible for coagulation, the values obtained with both of them ought to hav~ beo.?n almost the same. The colloidal particl~ are, however, acting as coagulating ions. Hence, in the case of the colloid, the coagulating power is the combined effect of the ferric ions and the colloidal particles. As the dialysis proceeds, the effect of the ferric ions continually diminishes. The ind1vidual effect of the ferric ions alone may be take~ to be equal to the effect of the .. equilibrium dialysate" as the concentration of the ferric ions in both of them is nearly the same. As the "equilibrium dialysate", obtained after the fifth stage of dialysis, ceases to coagulate the antimony sulphide sol, the values obtained for the colloid after this period may be taken to be the effect of the colloid particles alone. As the size of the colloid particles, as compared with the ferri~ chloride molecules, is very 458 P. M. UARVE AND A. 1<. J.\\lBO'fKAR FIG. 3 Coagulatiou va!l•e of tlu: colloid of dialysate aud cat. s{>cctt of colloid. liS ·= "6 ... X t:' - ~ I / r I l I I l r I r .. \ \ I I I I I I I I I 1 I -·---·-c ..... .s,. .. ....,....__,._r .... ~ ........._.._., • ·P....uf4•.1 ' \ ., .... ' ' '"', ~~ 7 8 SlaJ!I~S of equlllbrtotm. large, numerous ferric chloride molecules must ba.ve ttndergoue hydrolysis to build the colloidal 1enic hydroxide particle. The number of colloid particles so formed will naturally be extremely small in comparison with the n'umber of ferric ions which are used up. In spite of small numbe1, colloidal ferric hydroxide particles are highly effective in coagulation. They might therefore be functioning as multivalent coagulating ions with a valency many times more than three It should, however, be remembered that the process of mul ual coagulation is necessarily much more complicated and it is assumed only for the sake of simplicity, that the effect of the colloid and the intermicell· ary liquid is purely additive .. Stages of equilfbriJim. They might therefore be functioning as multivalent coagulating ions with a valency many times more than three It should, however, be remembered that the process of mul ual coagulation is necessarily much more complicated and it is assumed only for the sake of simplicity, that the effect of the colloid and the intermicell· ary liquid is purely additive .. after the fifth state of dialysis, it is found that the charge on the colloid decreases and the amount of the colloidal solution required for coagulating the same amount of the antimony sulphide sol i!;lcreases. This is what one would expect on theoretical grounds, as both the charge and the concentration of the ferric ions are decreasing. The constitution of the antimony sulphide sol r~mains unchanged as it is not subjected to dialysis, and hence, the effect of the antimony !"!Ulphide sol on the colloidal ierric hydroxide may be tak~p to be· constant. It should, however, be emphasized that the process of coagulation being not so simple, the difference between the two values is not necessarily an exact indication of the effcc·t of colloidal particles alone. It is qaite sufficent though to give one a clear idea about the influence of colloidal ferric hydroxide particles in mutual coagttlation. DEP.\RTMIN'l 01 CHEMIStRY, Wr.UOlf eor.!,.IGJI, Do!I.IBAY. DEP.\RTMIN'l 01 CHEMIStRY, Wr.UOlf eor.!,.IGJI, Do!I.IBAY. DEP.\RTMIN'l 01 CHEMIStRY, Wr.UOlf eor.!,.IGJI, Do!I.IBAY.
https://openalex.org/W2121064200	https://europepmc.org/articles/pmc3684536?pdf=render	English	null	Do family physicians advise younger people on cardiovascular disease prevention? A cross-sectional study from Slovenia	BMC family practice	2,013	cc-by	7,285	Abstract Background: One of the main family practice interventions in the younger healthy population is advice on how to keep or develop a healthy lifestyle. In this study we explored the level of counselling regarding healthy lifestyle by family physicians and the factors associated with it. Methods: A cross-sectional study with a random sample of 36 family practices, stratified by size and location. Each practice included up to 40 people aged 18–45 with low/medium risk for cardiovascular disease (CVD). Data were obtained by patient and practice questionnaires and semi-structured interviews. Several predictors on the patient and practice level for received advice in seven areas of CVD prevention were applied in corresponding models using a two-level logistic regression analysis. Results: Less than half of the eligible people received advice for the presented risk factors and the majority of them found it useful. Practices with medium patient list-sizes showed consistently higher level of advice in all areas of CVD prevention. Independent predictors for receiving advice on cholesterol management were patients’ higher weight (regression coefficient 0.04, p=0.03), urban location of practice (regression coefficient 0.92, p=0.04), organisation of education by the practice (regression coefficient 0.47, p=0.01) and practice list size (regression coefficient 6.04, p=0.04). Patients who self-assessed their health poorly more frequently received advice on smoking (regression coefficient −0.26, p=0.03). Hypertensive patients received written information more often (regression coefficient 0.66, p=0.04). People with increased weight more often received advice for children’s lifestyle (regression coefficient 0.06, p=0.03). We did not find associations with patient or practice characteristics and advice regarding weight and physical activity. We did not find a common pattern of predictors for advice. Conclusions: Counselling for risk diseases such as increased cholesterol is more frequently provided than basic lifestyle counselling. We found some doctors and practice factors associated with counselling behaviour, but the majority has to be explained by further studies. Keywords: Primary prevention, Cardiovascular diseases, Family practice, Counselling, Multilevel analysis still not unanimous [2] and is the subject of extensive re- search activity [3]. Petek et al. BMC Family Practice 2013, 14:82 Petek et al. BMC Family Practice 2013, 14:82 Petek et al. BMC Family Practice 2013, 14:82 http://www.biomedcentral.com/1471-2296/14/82 Open Access Do family physicians advise younger people on cardiovascular disease prevention? A cross-sectional study from Slovenia Davorina Petek1, Rok Platinovsek2, Zalika Klemenc-Ketis1,3 and Janko Kersnik1,3 Davorina Petek1, Rok Platinovsek2, Zalika Klemenc-Ketis1,3 and Janko Kersnik1,3 * Correspondence: davorina.petek@gmail.com 1Department of Family Medicine, Medical Faculty, University of Ljubljana, Poljanski nasip 58, 1000, Ljubljana, Slovenia Full list of author information is available at the end of the article © 2013 Petek 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. Subjects A random sample of 36 family medicine practices, strati- fied by size (small: up to two full-time equivalent (FTE) working family physicians on the same location, large: more than two FTE family physicians) and location (urban: more than 30,000 habitants, rural: 30,000 habi- tants or less) were included in the study. Out of 56 in- vited practices, 36 agreed to participate (response rate of 64.3%). According to European guidelines [4], primary preven- tion in the broad sense of counselling to retain a healthy lifestyle and improve risky behaviour should also be di- rected at healthy people without any known risk factors. This is a feature of the holistic and comprehensive ap- proach typical for family medicine. Furthermore, advice on healthy lifestyle cannot be given without assessing one’s lifestyle first. Therefore, GPs should advise all pa- tients on a healthy lifestyle - regardless of their health status and the presence of risk factors. In each practice, we aimed to include a random sam- ple of 40 people (age 18 to 45) from their list of regis- tered patients. Eligible people were those without any chronic cardiovascular diseases. Patients with a diag- nosed arterial hypertension or hypercholesterolemia but not assessed by the family physician as being at high risk for cardiovascular diseases were eligible for inclusion as well. Cardiovascular risk assessment was defined by using the Framingham score system, which is by na- tional agreement a compulsory tool for CVD risk assess- ment in the country. It provides scores from 0 to over 40 on the basis of patients’ age, sex, smoking history, systolic blood pressure and cholesterol levels. The scores are calculated automatically by a computer programme as part of each patient’s record keeping system after en- tering these data. Patients with scores from 0 to 20 on a Framingham scale were eligible for inclusion. It is mostly useful in age groups over 40. People were invited to take part in the study by phone and mail. An introductory letter, the letter of agreement to participate in the study, signed by their physician and practicing nurse were sent out. For the younger population some national guidelines even suggest to intervene at a lower risk threshold [5]. Despite recommendations, many physicians feel that healthcare’s resources and budget are insufficient for all primary prevention activities and cannot meet the needs of this group of people [6]. Methods been focused on the prevention of transmissible dis- eases and was typically provided through public health agencies. In recent years, cardiovascular prevention also became the task of family physicians who can provide individual counselling and use other strategies for lifestyle modification. European guidelines on car- diovascular prevention emphasise objectives of cardio- vascular prevention in the younger population: to retain a healthy lifestyle and to improve risky beha- viour where necessary [4]. The study was a part of the international European Practice Assessment of Cardiovascular risk manage- ment (Epa-Cardio) study, which involved nine European countries. Here, we present results of the Slovenian sample. The detailed description of methodology has already been published elsewhere [14]. Background Prevention of cardiovascular diseases for high risk popu- lation is a traditional task of family physicians as part of the comprehensive approach and community orientation [1]. There is ample evidence that multiple interventions lower mortality in high risk groups but the evidence for health-promoting activities in the general population is In primary cardiovascular diseases (CVD) preven- tion, a substantial part of the older generation is con- sidered to have high cardiovascular risk even with only a moderate elevation of modifiable risk factors. As a consequence, interventions are focused on this gener- ation and not on the younger population, for whom cardiovascular risk is also assessed less often. Primary prevention of the younger population has traditionally * Correspondence: davorina.petek@gmail.com 1Department of Family Medicine, Medical Faculty, University of Ljubljana, Poljanski nasip 58, 1000, Ljubljana, Slovenia Full list of author information is available at the end of the article Page 2 of 9 Petek et al. BMC Family Practice 2013, 14:82 http://www.biomedcentral.com/1471-2296/14/82 Petek et al. BMC Family Practice 2013, 14:82 http://www.biomedcentral.com/1471-2296/14/82 Subjects Many studies on cardiovascular prevention have focused on the population older than 40 or 50 years [7,8] or on a wide age range population [9] and have dealt with their risk assessment. Only a few studies have addressed cardio- vascular prevention for young people [10]. Attitudes of people towards lifestyle change, the role of their family doctor [11], patient expectations and received counselling [12] have been assessed in the whole population, not tak- ing into account any age differences. Younger groups of people usually come for a consult- ation when they need a health service for acute health problem and are classified as less frequent visitors. Young people are usually not included in routine pre- ventive programs. Slovenia is one of the few European countries that launched a National Programme of Car- diovascular Prevention already in 2001. One of its main critiques was that eligible people groups were too old (men 35–65, women 45–70 years old) in terms of long- term primary prevention impact. Out of 1,440 invited people, 953 of them returned the questionnaires. Later on we excluded 16 people because of missing data and performed a final analysis on 937 questionnaires by list-wise exclusion (response rate of 65.0%). Attitudes and practice toward preventive activities dif- fer also between the family physicians themselves; they depend on their own lifestyle [13] and on their practice characteristics such as a heavy workload. Questionnaire h ll d Each enrolled participant filled in the questionnaire that consisted of basic demographic data (gender, age, educa- tion, marital and employment status), self-assessment of health (using a five-point Likert-type scale ranging from excellent (5 points) to poor (1 point), length of attach- ment to the practice and frequency of yearly attendance of the practice. In the second part of the questionnaire, the participants stated whether they received advice on In our study, we aimed to explore the level of counsel- ling that young people receive in family medicine prac- tices and their experience with the received counselling, and to determine factors that can influence healthy life- style counselling provision. Petek et al. BMC Family Practice 2013, 14:82 http://www.biomedcentral.com/1471-2296/14/82 Page 3 of 9 risk factors for CVD and lifestyle and gave their opinion on the usefulness of this advice. Some questions on whether CVD advice was received were not administered to all respondents but rather to a smaller subgroup. The question on smoking advice was only administered to smokers, ex-smokers or occasional smokers and the ques- tion on children’s lifestyle was only administered to re- spondents with children. All patients also filled out the questionnaire on their lifestyle. The following validated questionnaires were used for this purpose: physical ac- tivity (RAPA questionnaire) [15], eating habits [16,17] and smoking status (MID-SIZED Model questionnaire) [18]. lifestyle more often than the rest of the population. A corresponding dummy-variable was included in the analyses. The software used to perform the estimation was the package lme4 [21] for R (R Development Core Team 2011) [22]. Statistical methods 312 respondents (33.3%) were overweight (BMI 25–30 kg/m2) and 138 (14.7%) were obese (BMI >30 kg/m2). 89 (9.5%) patients claimed that they had hypertension and 87 (9.3%) patients stated that they had hypercholesterolemia. The Epa-Cardio data cannot be analyzed with classical methods because these methods assume that the units were sampled independently. This assumption is violated in our case because people were sampled in clusters (practices). People attending the same practice are likely to be more similar than people attending different prac- tices. This clustering requires the use of multilevel ana- lysis [19,20], which allows for correct estimation of the standard errors of predictors in the explanatory model. This is especially important since we have a number of predictors that are measured at the practice level whose standard errors would be grossly underestimated with classical methods of analysis. The statistical analysis is focused on whether the participant received advice in one of the seven cardiovascular diseases prevention areas. Because the response was dichotomous (advice received/not received), we applied a two-level logistic re- gression to the data. We fit the so-called random inter- cept model seven times – to each of the areas of advice that the people could receive. The response variables and patient-level predictors are described in the follow- ing section. A specific part of our sample - males be- tween 35 and 45 years are included in the Slovenian National Preventive Programme, which is why we hy- pothesized that this group would receive advice on 424 (45.2%) of respondents stated that they never smoked cigarettes; others were current, occassional or past smokers. Lifestyle/ risk factors of the respondents According to the RAPA questionnaire, less than half of the sample of 850 people had adequate aerobic physical activity (384 respondents, 45.2%) and an even lower per- cent of the people were also performing exercises for stretching and for muscular strength (240 respondents, 28.5% of the sample). The average score on the REAP-S questionnaire for healthy diets of the respondents was 26.5 (SD=4.0). The lowest possible score on this item was 12 points and the highest score 39 with higher scores indicating a health- ier diet. The study was approved by the Slovenian National Committee on Medical Ethics (No. 87/11/07). Description of the sample The majority (23 or 63.9%) of the participating practices were small practices (employed no more than two GPs) and located in rural areas (26 practices or 72.2%). The mean age of people was 35.2 (SD 8,1) years. The basic demographic structure of the participating people is given in Table 1. Family physicians from each included practice filled out the questionnaire on practice characteristics. Additionally, the main researcher (DP) performed a semi-structured interview with all of the physicians. The questionnaire in- cluded questions on practice-led contacts for prevention (system for recalling people for CVD prevention), clinical information system (computer-supported patient file sys- tem), case finding methods to detect people with cardio- vascular risk factors, existing procedures for smoking cessation, work in community (participation of physicians and nurses in public healthcare programmes on lifestyle), education of GPs and nurses on CVD in the last year and registered patients’ list-size. Advice on lifestyle Figure 1 shows how many respondents recall being given advice in seven CVD areas. Less than half of the eligible respondents received advice for the presented risk fac- tors. In the two cases where the subgroups of (ex) smokers/occasional smokers and people with children were analysed, the percentages in Figure 1 pertain to the narrower subgroup as is also reflected in the lower num- ber of cases. The questionnaire also inquired whether the respon- dents found the advice to be helpful or not. Because the great majority of respondents reported the advice to be helpful, we performed the analysis by merging the cat- egories “not helpful” and “helpful” and performed the multilevel analyses on the resulting dichotomous (rather that multinomial) variable discriminating only between whether advice was given or not. Page 4 of 9 Petek et al. BMC Family Practice 2013, 14:82 http://www.biomedcentral.com/1471-2296/14/82 Table 1 Demographic, behavioural and health self- assessment characteristics of 937 patients aged from 18-45 years with low/medium cardiovascular risk Demographic characteristics No. (%) of patients Gender Men 384 (41.0) Women 528 (56.4) Missing 25 (2.7) Age 18-30 266 (28.4) 31-40 388 (41.4) 41-45 261 (27.9) Missing 22 (2.3) Education Primary school or less 134 (14.3) Secondary school 453 (48.3) University 313 (33.4) Missing 37 (3.9) Employment status Unemployed 116 (12.4) Employed 788 (84.1) Missing 33 (3.5) Marital status Married, cohabiting 649 (69.3) Single, divorced, widowed 264 (28.2) Missing 24 (2.6) Self assessment of health Poor 37 (3.9) Fair 139 (14.8) Good 386 (41.2) Very good 276 (29.5) Excellent 71 (7.6) Missing 28 (3.0) Practice attendance (years) ≤2 112 (12.0) 3-7 226 (24.1) 8-12 169 (18.0) >13 401 (42.8) Missing 29 (3.1) Visit frequency/year (n=918) 0-1 212 (22.6) 2-3 384 (41.0) 4-5 177 (18.9) 6-7 68 (7.3) ≥8 77 (8.2) Missing 19 (2.0) Table 1 Demographic, behavioural and health self- assessment characteristics of 937 patients aged from 18-45 years with low/medium cardiovascular risk Demographic characteristics No. Factors related to received advice for prevention of CVD Factors related to received advice for prevention of CVD Table 2 shows association of several predictor variables on patient and practice level with received preventive advice. We could not find any statistical prediction of the following variables on received CVD advice: gender, employment, marital status, practice size, case-finding methods, community resources. Some variables were borderline statistically significant. Advice on lifestyle (%) of patients Gender Men 384 (41.0) Women 528 (56.4) Missing 25 (2.7) Age 18-30 266 (28.4) 31-40 388 (41.4) 41-45 261 (27.9) Missing 22 (2.3) Education Primary school or less 134 (14.3) Secondary school 453 (48.3) University 313 (33.4) Missing 37 (3.9) Employment status Unemployed 116 (12.4) Employed 788 (84.1) Missing 33 (3.5) Marital status Married, cohabiting 649 (69.3) Single, divorced, widowed 264 (28.2) Missing 24 (2.6) Self assessment of health Poor 37 (3.9) Fair 139 (14.8) Good 386 (41.2) Very good 276 (29.5) Excellent 71 (7.6) Missing 28 (3.0) Practice attendance (years) ≤2 112 (12.0) 3-7 226 (24.1) 8-12 169 (18.0) >13 401 (42.8) Missing 29 (3.1) Visit frequency/year (n=918) 0-1 212 (22.6) 2-3 384 (41.0) 4-5 177 (18.9) 6-7 68 (7.3) Table 1 Demographic, behavioural and health self- assessment characteristics of 937 patients aged from 18-45 years with low/medium cardiovascular risk While conducting preliminary bivariate analyses, we encountered a curvilinear association between the pa- tient list size (the total number of patients on the prac- tice list of one GP) and the proportion of respondents who reported receiving CVD advice. Figures 2 and 3 de- pict the proportion of respondents who received advice as a function of the list size categorized into four cat- egories. The proportion of respondents who received ad- vice is lowest in the high and low numbers of people on the list, while this proportion is higher in the middle two categories of the list size. This trend is remarkably con- sistent across all eight areas of CVD advice. Summary of main findings and their contextualisation Figure 1 Advice and avowed helpfulness of advice- valid row percentages in the sample of younger people in national part of the Epa-Cardio study. Legend: Number of given advice in different areas differs due to missing values in the questionnaire and due to analysis of two subgroups of patients (smokers/ex-smokers, people with children). list size. With only one exception (advice on cigarette smoking in respondents with poor self-assessment of health), we did not find any meaningful (or the signifi- cance was borderline) association with patient/practice characteristics and advice in three models: for cigarette smoking, weight and physical activity. More often, provision of advice on healthy lifestyle for respondents who assessed their health worse was also found in other studies, athough the results were not consistent across all types of advice [26]. On the contrary to our results, other studies found that a rural location was associated with better provision of preventive services [32]. information possibilities. Another less often provided ad- vice was the advice on children’s lifestyle. In Slovenia, paediatricians are included in primary care and serve as children’s personal doctors. Regardless of this, family physicians should advise their patients – parents about children’s lifestyle too. Similarly, advice on community resources was not common, despite the fact that our country has a long tradition in community organisation of healthcare [30]. The percentage of advice on smoking was also surprisingly low, a classical field for education on healthy lifestyle. It was also perceived as the least helpful by respondents. We found it very interesting to receive a stable result in the association of middle-sized patient lists with more regularly given advice for all types of healthy living life- styles. Large list sizes of registered patients are an obs- tacle for preventive work, especially counselling [31]. Small list size practices are difficult to analyse, but they might experience difficulties in the organisation of pre- ventive activities as the doctors from such practices might have been involved in other primary care services or do not work full-time in the practice. Surprisingly, in the subsample of males from 35 to 45 years old, who are included in the Slovenian National Preventive Programme, our analyses could not confirm any higher figures on counselling for this group of people. Summary of main findings and their contextualisation Summary of main findings and their contextualisation The lifestyle of the studied younger family practice population showed disappointing results regarding the level of physical activity, healthy food habits, the num- ber of people who are currently regular or occasional smokers or who were smokers in the past and increased body weight/obesity. The figures on obesity were similar to the figures in a national epidemiological study, while the percentage of people exercising adequately showed a lower level of physical activity in our study [23]. In a group of healthy people 18–45 years of age (around 10% having isolated health problems like arterial hypertension or hypercholesterolemia), advice on any as- pect of cardiovascular prevention was given in only 50% of the people. Similar to other studies [24], we found that advice on physical exercise was the most common advice given (49.1%). Complex counselling on more aspects of life- style was also relatively common (45.3%). Percentages found in our study are slightly higher than in most of the other studies, where the figures in these studies were low in the early nineties [25], and higher in recent years [24] and varied considerably – advice on diet around 40% [26-28], on physical exercise 25-42% [26,27] and on smoking 30% or 40% for the adult population [27,29]. On the other hand, we found many »missing oppor- tunities«, such as a low level of given written informa- tion or advice to visit educational websites. The latter might be especially important because this was a young generation, which is familiar with the internet and its Page 5 of 9 Petek et al. BMC Family Practice 2013, 14:82 http://www.biomedcentral.com/1471-2296/14/82 education: children's lifestyle (n=592) local sports club or physical exercise (n=862) educational websites (n=859) written information on life style (n=866) two or more consultations on lifestyle (n=855) children's lifestyle (n=552) cholesterol (n=858) blood preassure (n=865) physical exercise/activity (n=858) weight (n=859) smoking (n=513) valid percent 0 20 40 60 80 100 no advice not helpful helpful Figure 1 Advice and avowed helpfulness of advice- valid row percentages in the sample of younger people in national part of the Epa-Cardio study. Legend: Number of given advice in different areas differs due to missing values in the questionnaire and due to analysis of two subgroups of patients (smokers/ex-smokers, people with children). Discussion on methodology The methodology has already been tested in the pilot study for the EPA-Cardio study. The questionnaire for participants contained questions, which were the results of previous work in the EPA-Cardio study [34]. Validated questionnaires were used to obtain information on diet, physical activity and smoking status. Questions on prac- tice characteristics were based on previous research on practice assessment [35]. A further strength of this study is a large sample, a good response rate and multi-level statistical analyses. The response rate was actually one of the highest com- pared to other countries in the Epa-Cardio study. The inclusion of the practices followed strict stratifica- tion rules and every practice that refused to participate was replaced by a practice from the same stratification field. This in fact could be a source of selection bias. Figure 2 Proportion of advice on smoking, blood pressure, physical activity and children’s lifestyle according to patient list size. As we can observe in other studies, the number of women in the sample was substantially higher, which can be attributed to the effect of self-choice: women are more frequent attenders of the practice than men [36] and more interested in participation in the survey than men [37]. Most of the respondents were long-term patients in the same practice, which is also in line with other European practices that have a patient list system [38]. The education of providers on preventive activities in- creased counselling on cholesterol. The result is interest- ing because it can show that education is too concentrated on secondary prevention and the management of risk dis- eases for CVD and does not emphasize lifestyle modifica- tion in general. We can compare our results with the systematic review of Bock et al. [33]: some practice characteristics that were related to higher levels of counselling in the review were the size of the practice, protocols for prevention and We also have to address problems of multiple testing. The statistical models include many predictor variables: nine on the patient level and nine on the practice level. We do not, however, consider multiple testing to be an important issue as the inclusion of each predictor was the- oretically well-grounded as described in the introduction. Summary of main findings and their contextualisation This goes in line with other studies – the group of younger people with low risk of cardiovascular disease is known to be rather neglected in the provision of pre- ventive activities [10,26]. We could not prove the positive relationship of elec- tronic support system on advice; on the opposite, prac- tices with computer-supported patient file systems gave less advice on several topics of lifestyle; we cannot ex- plain the result but it can be connected with the lack of systematic recording in e-patient files. Independent determinants for provided advice on managing blood cholesterol levels were the most numer- ous: patients’ weight and the following practice charac- teristics: urban location, practice education and patient Petek et al. BMC Family Practice 2013, 14:82 http://www.biomedcentral.com/1471-2296/14/82 Page 6 of 9 Petek et al. BMC Family Practice 2013, 14:82 proportion 0 0.2 0.4 0.6 list size (categorized) −1500 1500−2000 2000−2500 2500+ phisical activity smoking blood preassure children's lifestyle Figure 2 Proportion of advice on smoking, blood pressure, physical activity and children’s lifestyle according to patient list size. printed materials in the practice. The first one was proven also in our study. proportion 0 0.2 0.4 0.6 list size (categorized) −1500 1500−2000 2000−2500 2500+ phisical activity smoking blood preassure children's lifestyle Discussion on methodology proportion 0 0.2 0.4 0.6 list size (categorized) −1500 1500−2000 2000−2500 2500+ lifestyle weight cholesterol written information Figure 3 Proportion of complex lifestyle advice, advice on weight, cholesterol and given written information according to patient list size. y g The information on received advice came from the respondents and was not compared with their record and was therefore prone to subjectivity. We also have to be aware of possible social desirability in assessing the helpfulness of advice. However, other studies used the same methodology [39,40]. We also know that re- cords on advice are not very consistent either [41], and that the agreement between self-reports and medical records varies [42,43]. The delivery and recording of advice was much higher in the systems with adequate incentives, such as in the Quality and Outcomes Frame- work programme in the United Kingdom [44,45]. In Slovenian paper or electronic records, counselling has no structured recording form and we believe that re- cording is forgotten on many occasions, although we do not have any statistical data to prove it. No incentives are given for the recording of counselling in this age group. Moreover, respondents’ information/perception of received advice is the information that we were looking for in this study. Figure 3 Proportion of complex lifestyle advice, advice on weight, cholesterol and given written information according to patient list size. Petek et al. Discussion on methodology BMC Family Practice 2013, 14:82 http://www.biomedcentral.com/1471-2296/14/82 Page 7 of 9 Page 7 of 9 Table 2 Multilevel logistic regression analysis of patient and practice factors on received advice for prevention of CVD in the sample of younger people (regression coefficient above; statistical significance below in each cell) Smoking Weight Physical activity Cholesterol Lifestyle Children lifestyle Written in N= 478 N=775 N=779 N=770 N=777 N=501 N=774 intercept −3.89 -3.52 -3.45 -9.69 -3.12 -20.56 -3.32 0.16 0.17 0.12 0.00 0.22 0.00 0.29 Gender (female) 0.36 -0.07 0.07 -0.01 -0.20 0.14 0.23 0.11 0.68 0.67 0.98 0.26 0.60 0.27 age -0.01 0.01 0.00 -0.01 0.01 -0.06 -0.02 0.75 0.29 0.89 0.70 0.21 0.01 0.19 Male 35-45 years 1.42 1.17 0.81 -1.08 -0.04 1.04 -0.36 0.05 0.05 0.14 0.23 0.95 0.66 0.05 education -0.03 0.08 0.04 0.27 -0.15 -0.06 -0.20 0.87 0.54 0.77 0.09 0.25 0.74 0.09 Employment (not employed) 0.14 0.05 -0.07 0.46 0.34 -0.10 0.36 0.65 0.85 0.76 0.10 0.17 0.81 0.10 Marital status (not married)1 '0.06 '0.01 0.00 0.09 -0.11 -0.56 -0.15 0.82 0.97 0.99 0.71 0.58 0.07 0.51 Self assessment of health2 -0.26 -0.08 0.00 -0.06 0.14 0.11 0.08 0.03 0.36 0.96 0.60 0.12 0.47 0.47 BMI3 0.03 0.01 0.00 0.04 -0.01 0.06 0.03 0.14 0.53 0.99 0.03 0.47 0.03 0.15 Hypertension4 0.16 0.20 -0.19 -0.08 0.32 0.37 0.66 0.67 0.50 0.50 0.82 0.27 0.38 0.04 Practice size (large) -0.03 -0.04 0.01 0.06 0.33 0.04 0.00 0.93 0.89 0.97 0.87 0.33 0.94 1.00 Practice loca- tion (urban) -0.04 -0.21 -0.13 0.92 -0.18 -0.02 0.41 0.91 0.60 0.71 0.04 0.66 0.96 0.42 Recall system CVD5 -0.74 -0.93 -0.53 -0.76 -0.34 0.33 -0.60 0.14 0.05 0.22 0.16 0.49 0.61 0.32 E pt file6 -0.48 -0.48 -0.75 -2.14 -1.29 -1.09 -0.92 0.27 0.26 0.05 0.00 0.00 0.06 0.09 Case finding methods7 0.21 0.25 0.20 0.00 0.29 -1.19 0.84 0.73 0.67 0.70 1.00 0.62 0.15 0.29 Community resources8 -0.06 -0.21 -0.07 -0.13 -0.07 -0.05 0.01 0.70 0.15 0.58 0.42 0.63 0.82 0.96 Education organisation9 -0.03 0.10 0.22 0.47 0.12 0.00 0.09 0.88 0.55 0.14 0.01 0.47 0.99 0.67 List size10 linear 3.99 3.16 2.99 6.04 2.44 21.21 1.19 0.12 0.20 0.15 0.04 0.31 0.00 0.70 Table 2 Multilevel logistic regression analysis of patient and practice factors on received advice for prevention of CVD in the sample of younger people (regression coefficient above; statistical significance below in each cell) Petek et al. References 1. Wonca Europe: The European Definition of Primary Care. http://www. woncaeurope.org/gp-definitions. 1. Wonca Europe: The European Definition of Primary Care. http://www. woncaeurope.org/gp-definitions. Discussion on methodology BMC Family Practice 2013, 14:82 http://www.biomedcentral.com/1471-2296/14/82 Page 8 of 9 Table 2 Multilevel logistic regression analysis of patient and practice factors on received advice for prevention of CVD in the sample of younger people (regression coefficient above; statistical significance below in each cell) (Continued) Table 2 Multilevel logistic regression analysis of patient and practice factors on received advice for prevention of CVD in the sample of younger people (regression coefficient above; statistical significance below in each cell) (Continued) List size quadratic -0.88 -0.72 -0.62 -1.19 -0.46 -5.30 -0.24 0.13 0.19 0.19 0.07 0.40 0.01 0.72 Legend 1Marital status: 1= Married / cohabiting, 2= Single, Separated / divorced, Widowed. 2Self-assessment of health: How would you estimate your health status in general: 1= excellent, 2= very good and 3= good were aggregated to “good”, 4= fair and 5= poor to “poor”. 3Value of BMI (kg/m2). 4Yes/No answer of the patient if he/she has hypertension. 5Use of system for recall of people at risk for CVD. 6Computer-supported patient file system. 7If the practice uses case-finding methods to detect people with cardiovascular risk factors. 8Do practice physicians and nurses cooperate in local/community campaigns or actions? 9Education of providers: education of all nurses/GPs in the last five years on CVD, education of at least one nurse/GP on CVD in the last 15 months. 10Number of people on the patient list. As this was a cross-sectional study, we could not as- sess the effect of the advice given. We also did not in- clude advice on alcohol consumption in the study. Funding for the international EPA-Cardio study was supported by the Bertelsmann foundation (http://www.bertelsmann-stiftung.de/cps/rde/xchg/ bst/hs.xsl/prj_8519.htm), which had no involvement in the study design, analyses or interpretation of the data. In Slovenia it was supported also by the Slovenian Family Medicine Society. Funding for the international EPA-Cardio study was supported by the Bertelsmann foundation (http://www.bertelsmann-stiftung.de/cps/rde/xchg/ bst/hs.xsl/prj_8519.htm), which had no involvement in the study design, analyses or interpretation of the data. In Slovenia it was supported also by the Slovenian Family Medicine Society. Authors’ contributions DP i i d i h d y 8. Guallar E, Banegas JR, Blasco-Colmenares E, Jiménez FJ, Dallongeville J, Halcox JP, et al: Excess risk attributable to traditional cardiovascular risk factors in clinical practice settings across Europe - The EURIKA Study. BMC Publ Health 2011, 11:704. 8. Guallar E, Banegas JR, Blasco-Colmenares E, Jiménez FJ, Dallongeville J, Halcox JP, et al: Excess risk attributable to traditional cardiovascular risk factors in clinical practice settings across Europe - The EURIKA Study. BMC Publ Health 2011, 11:704. DP participated in the design of the study, acquisition and interpretation of the data and drafted the manuscript. RP performed the statistical analysis and interpretation and participated in manuscript writing. ZKK participated in the acquisition of the data and manuscript writing. JK coordinated the study, participated in the acquisition and interpretation of the data and manuscript writing. All authors read and approved the final manuscript. 9. Thio SL, Twickler TB, Cramer MJ, Giral P: National differences in screening programmes for cardiovascular risks could obstruct understanding of cardiovascular prevention studies in Europe. Neth Heart J 2011, 19:458–463. Conclusion In our study we could relate some people and practice characteristics to certain forms of cardiovascular preven- tion advice provided. It seems that basic counselling for lifestyle (smoking, weight and physical activity) is not provided widely enough to the younger population and we could prove only some associations in each model. It seems that the providers’ education is still too concen- trated on what they perceive as a disease. The recording of advice in electronic files needs to be standardized and carried out systematically. Policy makers should be aware that list size matters in the uptake of preventive activities. 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Also, there is a need for larger international studies in this subject, which will use prospective methodology to assess the success of such advice. 1Department of Family Medicine, Medical Faculty, University of Ljubljana, Poljanski nasip 58, 1000, Ljubljana, Slovenia. 2Faculty of Social Sciences, University of Ljubljana, Kardeljeva ploscad 5, 1000, Ljubljana, Slovenia. 3Department of Family Medicine, Medical Faculty, University of Maribor, Slomskov trg 15, 2000, Maribor, Slovenia. Acknowledgments Topolski TD, LoGerfo J, Patrick DL, Williams B, Walwick J, Patrick MB: The rapid assessment of physical activity (RAPA) among older adults. Prev Chronic Dis 2006, 3:A118. http://www.ncbi.nlm.nih.gov/pmc/articles/ PMC1779282/?tool=pubmed. 37. Diviak KR, Wahl SK, O’Keefe JJ, Mermelstein RJ, Flay BR: Recruitment and retention of adolescents in a smoking trajectory study: who participates and lessons learned. Subst Use Misuse 2006, 41:175–182. 38. 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Acknowledgments 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 doi:10.1186/1471-2296-14-82 Cite this article as: Petek et al.: Do family physicians advise younger people on cardiovascular disease prevention? A cross-sectional study from Slovenia. BMC Family Practice 2013 14:82. Petek et al. BMC Family Practice 2013, 14:82 http://www.biomedcentral.com/1471-2296/14/82 Submit your next manuscript to BioMed Central and take full advantage of: 30. Klancar D, Svab I, Kersnik J: Vizijaprihodnostizdravstvenihdomov v Sloveniji. The vision of health centers in Slovenia. ZdravVar 2010, 49:37–43. In slovenian. • Convenient online submission 31. Pelletier-Fleury N, Le Vaillant M, Hebbrecht G, Boisnault: Determinants of preventive services in general practice. A multilevel approach in cardiovascular domain and vaccination in France. Health Policy 2007, 81:218–227. 32. Laws RA, Jayasinghe UW, Harris MF, Williams AM, Powell Davies G, Kemp LA, Community Health SNAP Project Team: Explaining the variation in the management of lifestyle risk factors in primary health care: a multilevel cross sectional study. BMC Publ Health 2009, 9:165.
https://openalex.org/W4245946208	https://journals.plos.org/plosone/article/file?type=printable&id=10.1371/journal.pone.0074689	English	null	Correction: Body Sodium Overload Modulates the Firing Rate and Fos Immunoreactivity of Serotonergic Cells of Dorsal Raphe Nucleus	PloS one	2,014	cc-by	10,629	Abstract In order to determine whether serotonergic (5HT) dorsal raphe nucleus (DRN) cells are involved in body sodium status regulation, the effect of a s.c. infusion of either 2 M or 0.15 M NaCl on 5HT DRN neuron firing was studied using single unit extracellular recordings. In separate groups of 2 M and 0.15 M NaCl-infused rats, water intake, oxytocin (OT) plasma concentration, urine and plasma sodium and protein concentrations were also measured. Also, to determine the involvement of particular brain nuclei and neurochemical systems in body sodium overload (SO), animals from both groups were perfused for brain immunohistochemical detection of Fos, Fos-OT and Fos-5HT expression. SO produced a significant increase in serotonergic DRN neuron firing rate compared to baseline and 0.15 M NaCl-infused rats. As expected, 2 M NaCl s.c. infusion also induced a significant increase of water intake, diuresis and natriuresis, plasma sodium concentration and osmolality, even though plasma volume did not increase as indicated by changes in plasma protein concentration. The distribution of neurons along the forebrain and brainstem expressing Fos after SO showed the participation of the lamina terminalis, extended amygdala, supraoptic and paraventricular hypothalamic nuclei in the neural network that controls osmoregulatory responses. Both Fos-OT immunoreactive and plasma OT concentration increased after s.c. hypertonic sodium infusion. Finally, matching the ‘‘in vivo’’ electrophysiological study, SO doubled the number of Fos-5HT immunolabeled cells within the DRN. In summary, the results characterize the behavioral, renal and endocrine responses after body sodium overload without volume expansion and specify the cerebral nuclei that participate at different CNS levels in the control of these responses. The electrophysiological approach also allows us to determine in an ‘‘in vivo" model that DRN 5HT neurons increase their firing frequency during an increase in systemic sodium concentration and osmolality, possibly to modulate sodium and water intake/excretion and avoid extracellular volume expansion. Editor: Yvette Tache, University of California, Los Angeles, United States of America Received April 5, 2013; Accepted August 5, 2013; Published September 20, 2013 Copyright: 2013 Godino 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. Funding: This work was supported in part by grants from: SECyT grant 2010–2011. N: 05/1649; CONICET grant 2010–2012. PIP 2561; ANPCyT grant 2011–2014. Bicentenario, PICT-2010-2072 (Resolution Nu 098/11); MYNCyT Co´rdoba grant 2011–2013. Body Sodium Overload Modulates the Firing Rate and Fos Immunoreactivity of Serotonergic Cells of Dorsal Raphe Nucleus Andrea Godino, Soledad Pitra, Hugo F. Carrer, Laura Vivas Instituto de Investigacio´n Me´dica Mercedes y Martı´n Ferreyra (INIMEC-CONICET-Universidad Nacional de Co´rdoba), Co´rdoba, Argentina Andrea Godino, Soledad Pitra, Hugo F. Carrer, Laura Vivas Andrea Godino, Soledad Pitra, Hugo F. Carrer, Laura Vivas Instituto de Investigacio´n Me´dica Mercedes y Martı´n Ferreyra (INIMEC-CONICET-Universidad Nacional de Co´rdoba), Co´rdoba, Argentina Abstract PID (Resolution 000113/2011); Fundacio´n Roemmers grant to novel investigators. 2011–2013; ANPCyT grant 2012–2014 for young investigators. PICT 2011–2014 (Resolution 140/12); and ISN (International Society for Neurochemistry) Research supplies for use in the applicant’s home laboratory. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. E-mail: agodino@immf.uncor.edu September 2013 \| Volume 8 \| Issue 9 \| e74689 Editor: Yvette Tache, University of California, Los Angeles, United States of America Control of Sodium Balance by Serotonergic Neurons concentration of NaCl consumed, suggesting that this system is involved in the inhibition of sodium appetite under conditions of satiety [11]. Significant increased activity was also observed in serotonergic DRN cells after EVE [10]. Moreover, our connec- tional studies using a retrograde marker, Fluorogold, in combina- tion with Fos have demonstrated an important functional projection from the DRN to the LPBN and the LT, which are involved in different models of fluid and sodium balance regulation [12–14]. ing. This suggests that the serotonergic system within the DRN participates in the brain circuit that controls this homeostatic response. Oxytocinergic neural activity and OT plasma concen- tration also increased after sodium overload without EVE, confirming their involvement in this physiological condition. The central circuit activated after body sodium overload involves specific nuclei along the brainstem, lamina terminalis, hypotha- lamic and central extended amygdala areas. Ethical Statement y p [ ] Together these results suggest that body sodium levels modulate the activity of serotonergic DRN cells. However, their specific participation in a model of systemic hypernatremia/hyperosmo- larity without blood volume expansion, directly recording ‘‘in - vivo’’ DRN cell electrical activity during sodium overload, has not yet been studied. For this purpose, anesthetized animals were used, subjected to a single unit extracellular recording of serotonergic DRN cells and receiving a s.c. infusion of either 2 M or 0.15 M NaCl (0.6 ml/100 g b w) solution for one minute. The distribution of neurons along the forebrain and brainstem that express Fos, the double immunolabeling of Fos-5HT, and Fos-OT were also identified under this sodium overload model, and behavioral, renal and endocrine responses were characterized. All experimental procedures were approved and carried out in accordance with the guidelines of the Ethical Committee of the Instituto Ferreyra for the use and care of laboratory animals. The protocol designed to minimize animal suffering was approved by this Committee (Permit Number: PIP 2009/2011, 11220080102561). Materials and Methods In agreement with the above evidence, other authors have demonstrated that DRN lesion and 5HT antagonist (Methyser- gide) injection into the LPBN increased sodium intake induced by sodium depletion and decreased sodium and potassium renal excretion and endocrine responses (OT and AVP) after EVE. Similarly, the 5HT agonist (5HT2A/2C, 2,5-dimethoxy-4-io- doamphetamine hydrobromide), injected into the LPBN, in- creased the renal and endocrine response after EVE and inhibited sodium intake induced by different experimental models [15–20]. demonstrated that DRN lesion and 5HT antagonist (Methyser- gide) injection into the LPBN increased sodium intake induced by sodium depletion and decreased sodium and potassium renal excretion and endocrine responses (OT and AVP) after EVE. Similarly, the 5HT agonist (5HT2A/2C, 2,5-dimethoxy-4-io- doamphetamine hydrobromide), injected into the LPBN, in- creased the renal and endocrine response after EVE and inhibited sodium intake induced by different experimental models [15–20]. Together these results suggest that body sodium levels modulate the activity of serotonergic DRN cells. However, their specific participation in a model of systemic hypernatremia/hyperosmo- larity without blood volume expansion, directly recording ‘‘in - vivo’’ DRN cell electrical activity during sodium overload, has not yet been studied. For this purpose, anesthetized animals were used, subjected to a single unit extracellular recording of serotonergic DRN cells and receiving a s.c. infusion of either 2 M or 0.15 M NaCl (0.6 ml/100 g b w) solution for one minute. The distribution of neurons along the forebrain and brainstem that express Fos, the double immunolabeling of Fos-5HT, and Fos-OT were also identified under this sodium overload model, and behavioral, renal and endocrine responses were characterized. The experiments used adult, Wistar-derived male rats, born and reared in the breeding colony at the Instituto Ferreyra (INIMEC, Co´rdoba, Argentina). Animals weighing 250–300 g were housed singly in metabolic cages. Room lights were on for 12 h/day, and temperature was controlled at 23uC. Introduction brainstem level, this stimulus involved the activity of the lateral parabrachial nucleus (LPBN), locus coeruleus (LC), ventrolateral medulla (VL), nucleus of the solitary tract (NTS) and area postrema (AP). Together, all these areas work in coordination to mediate the autonomic, endocrine and behavioral responses inherent in osmoregulation [4,7,8]. Under normal volemia conditions, increased natremia or plasma osmolality involves compensatory responses such as water intake, sodium appetite inhibition, plasma release of oxytocin (OT) and vasopressin (AVP), and consequently renal sodium excretion and water retention [1]. The central circuit involved in the control of these behavioral and physiological responses induced by hypernatremia has been studied, analyzing the expression of brain immediate early genes [2–4]. Enhanced c-fos expression as shown by increased Fos immunoreactivity was identified within the magnocellular groups of OT and AVP hypothalamic paraventricular and supraoptic nuclei (PVN and SON), and matching the observed increase of both peptides in plasma [4,5]. The increased natremia and osmolality is centrally detected by the circumventricular organs of the lamina terminalis (CVOs of LT), which have osmo- and sodium- sensitive cells and have shown increased Fos immunoreactivity (Fos-ir) after different paradigms of body salt loading [4–6]. At Our previous results involved the serotonergic neurons of the dorsal raphe nucleus (DRN) in the regulation of fluid and electrolyte balance after particular hydroelectrolyte disturbances [9–14]. These studies analyzed the double immunoreactive cells for Fos and serotonin along the raphe system, before and after sodium intake induced by sodium depletion and after extracellular volume expansion (EVE). The data indicate that body sodium status modulates the activity of serotonergic DRN neurons, since Fos-5HT-ir decreased after sodium depletion and increased in animals in positive sodium balance or in process of reestablishing sodium balance. Serotonergic cells of the DRN were also activated after body sodium status was reestablished, independently of the September 2013 \| Volume 8 \| Issue 9 \| e74689 September 2013 \| Volume 8 \| Issue 9 \| e74689 1 PLOS ONE \| www.plosone.org Control of Sodium Balance by Serotonergic Neurons Sodium Overload (So) After 4 h of food and water deprivation, each adult rat was weighed, injected subcutaneously (s.c.) on the back with xilocaine (in an effort to reduce non-specific treatment- associated pain and stress), and 5 minutes later with hypertonic saline (2 M NaCl) delivered through a 26-gauge needle (0.6 ml/ 100 g body weight, for 1 minute) using an infusion pump (B. Braun, Germany), and then returned to its metabolic cage. Control rats were injected similarly but with isotonic saline Our electrophysiological study demonstrates that the firing rate of putative 5HT DRN neurons increases after body sodium overload, matching the pattern of Fos-5HT double immunostain- Figure 1. Effect of 2 M and 0.15 M NaCl sc infusions on firing frequency of putative 5HT-DRN neurons. Values are means of 1 minutes intervals from 2 M NaXl (n = 14, black circles) or 0.15 M NaCl (n = 16, white circles) groups, before and after NaCl infusion indicated by the arrow. Values are means 6 SE. P,0.05 vs. 0.15 M NaCl group+P,0.05 vs. basal recording. (2-way ANOVA post hoc LSD test). doi:10.1371/journal.pone.0074689.g001 Figure 1. Effect of 2 M and 0.15 M NaCl sc infusions on firing frequency of putative 5HT-DRN neurons. Values are means of 1 minutes intervals from 2 M NaXl (n = 14, black circles) or 0.15 M NaCl (n = 16, white circles) groups, before and after NaCl infusion indicated by the arrow. Values are means 6 SE. P,0.05 vs. 0.15 M NaCl group+P,0.05 vs. basal recording. (2-way ANOVA post hoc LSD test). doi:10.1371/journal.pone.0074689.g001 Figure 1. Effect of 2 M and 0.15 M NaCl sc infusions on firing frequency of putative 5HT-DRN neurons. Values are means of 1 minutes intervals from 2 M NaXl (n = 14, black circles) or 0.15 M NaCl (n = 16, white circles) groups, before and after NaCl infusion indicated by the arrow. Values are means 6 SE. P,0.05 vs. 0.15 M NaCl group+P,0.05 vs. basal recording. (2-way ANOVA post hoc LSD test). doi:10.1371/journal.pone.0074689.g001 September 2013 \| Volume 8 \| Issue 9 \| e74689 PLOS ONE \| www.plosone.org 2 Control of Sodium Balance by Serotonergic Neurons Figure 2. Histograms showing the effect of NaCl sc infusion on electrical activity of puta activity at basal, and after NaCl and fluoxetine infusion are also shown (mV vs ms). A)- 2 M NaCl sc doi:10.1371/journal.pone.0074689.g002 Figure 2. Control of Sodium Balance by Serotonergic Neurons The recording sites are represented within plates 48 and 50 from Paxinos and Watson (1997) corresponding with 27.64 mm (A) and 28.00 mm (B) distance from bregma, specifying the excitatory (+), inhibitory (2), control () and neutral (O) responses. DRD: dorsal subdivision of dorsal raphe nucleus, DRVL: ventrolateral subdivision of dorsal raphe nucleus, DRV: ventral subdivision of dorsal raphe nucleus, Aq: cerebral aqueduct. doi:10.1371/journal.pone.0074689.g003 Figure 3. Location of recording sites within the DRN from 2 M NaCl or 0.15 M NaCl infused rats. The recording sites are represented within plates 48 and 50 from Paxinos and Watson (1997) corresponding with 27.64 mm (A) and 28.00 mm (B) distance from bregma, specifying the excitatory (+), inhibitory (2), control () and neutral (O) responses. DRD: dorsal subdivision of dorsal raphe nucleus, DRVL: ventrolateral subdivision of dorsal raphe nucleus, DRV: ventral subdivision of dorsal raphe nucleus, Aq: cerebral aqueduct. doi:10.1371/journal.pone.0074689.g003 – Excitatory response: when firing frequency increases 0.2 Hz or more after infusion in comparison to baseline activity. – Inhibitory response: when firing frequency decreases 0.2 Hz or more after infusion in relation to baseline. – Neutral response: the absolute changes in firing frequency after infusion do not exceed 0.2 Hz in relation to baseline activity. (0.15 M NaCl). Individual rats from each group were used for urine and plasma measurements, OT radioimmunoassay or brain immunohistochemical detection of Fos, Fos-OT and Fos- 5HT expression as described below. (0.15 M NaCl). Individual rats from each group were used for urine and plasma measurements, OT radioimmunoassay or brain immunohistochemical detection of Fos, Fos-OT and Fos- 5HT expression as described below. For quantitative analysis, the registered putative 5HT cells were analyzed using a two-way ANOVA mixed with repeated measures (treatment as main factor and time as repeated measures) was used. Post-hoc comparisons were made with the least significant difference test. Another group of animals was used for the electrophysiological study, and were anesthetized and subjected to SO (as previously described) during the extracellular recording. Sodium Overload (So) Histograms showing the effect of NaCl sc infusion on electrical activity of putative 5HT-DRN neurons. Fragments recording activity at basal, and after NaCl and fluoxetine infusion are also shown (mV vs ms). A)- 2 M NaCl sc infusion, B)- 0.15 M NaCl sc infusion. doi:10.1371/journal.pone.0074689.g002 Figure 2. Histograms showing the effect of NaCl sc infusion on electrical activity of putative 5HT-DRN neurons. Fragments recording activity at basal, and after NaCl and fluoxetine infusion are also shown (mV vs ms). A)- 2 M NaCl sc infusion, B)- 0.15 M NaCl sc infusion. doi:10.1371/journal.pone.0074689.g002 September 2013 \| Volume 8 \| Issue 9 \| e74689 September 2013 \| Volume 8 \| Issue 9 \| e74689 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 3 Control of Sodium Balance by Serotonergic Neurons Control of Sodium Balance by Serotonergic Neurons Figure 3. Location of recording sites within the DRN from 2 M NaCl or 0.15 M NaCl infused rats. The recording sites are represented within plates 48 and 50 from Paxinos and Watson (1997) corresponding with 27.64 mm (A) and 28.00 mm (B) distance from bregma, specifying the excitatory (+), inhibitory (2), control () and neutral (O) responses. DRD: dorsal subdivision of dorsal raphe nucleus, DRVL: ventrolateral subdivision of dorsal raphe nucleus, DRV: ventral subdivision of dorsal raphe nucleus, Aq: cerebral aqueduct. doi:10.1371/journal.pone.0074689.g003 from cells which displayed a signal: noise ratio 2:1 or more. DRN serotonergic neurons were tentatively identified on the basis of characteristic firing patterns and pharmacological characterization for an ‘‘in vivo’’ or ‘‘in vitro’’ spontaneously active neurons described previously by Baraban & Aghajanian (1980), Kirby et al., (2003) and others [22–26]. These features includes: a slow (0.5–2.5 Hz), regular firing rate and biphasic action potentials of 1.0–3.0 ms duration, while pharmacological phenotype characterization was performed by 5HT1A stimulation induced by iv injection of fluoxetine (a serotonin reuptake inhibitor which causes a decrease in the firing frequency of serotonergic neurons mainly by 5HT1A autoreceptors stimulation). Thus, we only recorded the spontane- ously active neurons which have a slow and regular firing rate that are expected to be inhibited by 5HT1A autoreceptors stimulation [24]. Electrode potentials, which had been previously passed through a high-impedance amplifier, were passed through a window discriminator and screened on an audio amplifier. Integrated histograms generated by the analogy output of the window discriminator were analyzed on-line with a computer. Only one neuron was recorded per animal in order to analyze the response to 2 M NaCl or 0.15 M NaCl injection. The baseline activity of each neuron was recorded for 3–5 min before any treatment, and changes in neuronal firing were observed for 15 min after s.c. saline infusions (2 M or 0.15 M NaCl). The mean discharge rate was determined over 1 min intervals. Pharmaco- logical phenotype characterization was then performed by iv injection of fluoxetine. The recording site was marked by injecting Pontamine sky blue dye using a pneumatic pressure pump (Medical Systems Corp. NY MS-2). For the qualitative analysis, the following criteria were used to characterize the neuronal response to hypertonic or isotonic infusions: Figure 3. Location of recording sites within the DRN from 2 M NaCl or 0.15 M NaCl infused rats. Experiment 1: Analysis of the Firing Frequency of Serotonergic DRN Neurons during Sodium Overload A two-way ANOVA (treatment and time factors) was used for the analysis of plasma electrolytes, protein concentration and osmo- lality. Post-hoc comparisons were made with the least significant difference test. A t-test was used for the urinary electrolytes and osmolality after treatments. radioimmunoassay as described by Morris and Alexander (1989) [27]. For the assay, OT was extracted from 1 ml of plasma with acetone and petroleum ether. The percentage of recovery after extraction was 85%. The assay sensitivity and intra- and inter- assay coefficients of variation were 0.9 pg/ml, 7% and 12.6%. A two-way ANOVA was used for plasma oxytocin concentration analysis. radioimmunoassay as described by Morris and Alexander (1989) [27]. For the assay, OT was extracted from 1 ml of plasma with acetone and petroleum ether. The percentage of recovery after extraction was 85%. The assay sensitivity and intra- and inter- assay coefficients of variation were 0.9 pg/ml, 7% and 12.6%. A two-way ANOVA was used for plasma oxytocin concentration analysis. Experiment 1: Analysis of the Firing Frequency of Serotonergic DRN Neurons during Sodium Overload Water intake induced by SO. Food- and water-deprived animals were s.c. saline infused (2 M NaCl or 0.15 M NaCl), returned to metabolic cages without food and provided immediate access to water. Cumulative intake was measured in calibrated burettes at 0, 30, 60, 120, and 180 min. Drinking latencies were recorded immediately after saline infusion. A one-way repeated measures ANOVA was used for the analysis of water intake. Post- hoc comparisons were made with the least significant difference test. The rats were anesthetized with urethane 50% (0.3 ml/100 g b w) and then were catheterized in the jugular vein. Body temperature was maintained at 36–37uC, with a feedback- controlled heating pad. The animals were mounted onto a stereotaxic frame, their skulls exposed, and a hole was drilled above the DRN into which an electrode was lowered (7.8 mm posterior to bregma, 1.0 mm lateral to midline suture, 5.5– 7.0 mm below the dura mater) [21] by means of a hydraulic microdrive. The saggital sinus was ligated and displaced to avoid puncturing this sinus during the descent of the electrode. Single unit DRN discharge was recorded with glass micropipettes (1 mm tip diameter recording electrode, World Precision Instruments, Inc., item number 1B120F-4) filled with 2 M NaCl solution containing 2% Pontamine Sky Blue. The firing rate was obtained Determination of plasma OT concentration. For the plasma OT concentration assay, different groups of animals were used from those used in the immunohistochemical and electro- physiological studies. Animals were decapitated and bled before and 5, 10 and 15 minutes after SO. Trunk blood was collected in chilled plastic tubes containing heparin. Plasma OT level was measured by September 2013 \| Volume 8 \| Issue 9 \| e74689 PLOS ONE \| www.plosone.org 4 Control of Sodium Balance by Serotonergic Neurons Figure 4. Cumulative volume of water drunk during the intake test (3 h) after 2 M NaCl or 0.15 M infusion. Values are means 6 SE. P,0.05 vs. Control group (n = 9). doi:10.1371/journal.pone.0074689.g004 ume of water drunk during the intake test (3 h) after 2 M NaCl or 0.15 M infusion. Values are means 6 SE n = 9). 4689 g004 Figure 4. Cumulative volume of water drunk during the intake test (3 h) after 2 M NaCl or 0.15 M infusion. Values are means 6 SE. P,0.05 vs. Control group (n = 9). doi:10.1371/journal.pone.0074689.g004 were analyzed by vapor pressure osmometer (VAPRO 5520). Experiment 3: Brain Pattern of Fos, Fos-OT and Fos-5HT Immunoreactivity after SO Electrolytes and protein assays. To analyze the plasma electrolyte concentration in the 2 M NaCl or 0.15 M NaCl groups, animals were decapitated and bled before and 5, 10 and 15 minutes after SO. Samples were centrifuged and 1 ml of plasma was extracted and stored at 220uC. Urinary samples were taken after the intake test (180 min. after s.c. infusion) and immediately centrifuged, and 1 ml was extracted and stored at 220uC. Electrolyte concentrations of these samples were analyzed by flame photometry (Hitachi 911, automatic analyzer). Plasma volume was inferred from the plasma protein concentration measured according to Lowry [28]. Plasma and urine osmolality Animals were perfused 90 min after s.c. 2 M NaCl or 0.15 M NaCl infusion and their brains extracted for immunohistochemical detection of Fos, Fos-OT and Fos-5HT. For this purpose, the different groups of rats were anesthetized with thiopentone (100 mg/kg ip) and perfused transcardially with ,100 ml normal saline followed by ,400 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.2). The 90 minutes interval after stimulation was chosen in order to be able to compare the dium and protein concentration and plasma osmolality after 2 M NaCl or 0.15 M NaCl s.c. injection. Table 1. Plasma sodium and protein concentration and plasma osmolality after 2 M NaCl or 0.15 M NaCl s.c. injection. Time (min) Plasma Sodium Concentration meq/l Plasma Osmolality mosmol/KgH2O Plasma Protein Concentration g/dl NaCl 0.15 M NaCl 2 M NaCl 0.15 M NaCl 2 M NaCl 0.15 M NaCl 2 M 0 146.261.0 146.261.0 296.9160.55 296.9160.55 6.2360.28 6.2360. 28 5 145.161.2 148.461.1 287.8160.89 298.3860.68* 6.6260. 34 6.1460.30 10 144.561.1 151.461.0+ 298.0660.78 309.3560.61+ 6.4460.32 6.1060.28 15 142.761.2 148.261.1* 296.7860.89 303.1660.68+ 6.1860.34 6.3260.30 Values are means 6 SE; n = 5. P,0.05 Significantly different between NaCl 2 M and NaCl 0.15 M groups.+P,0.05 Significantly different from baseline levels (time 0). doi:10.1371/journal.pone.0074689.t001 ble 1. Plasma sodium and protein concentration and plasma osmolality after 2 M NaCl or 0.15 M NaCl Values are means 6 SE; n = 5. P,0.05 Significantly different between NaCl 2 M and NaCl 0.15 M groups.+P,0.05 Significantly different from baseline levels (time 0). doi:10.1371/journal.pone.0074689.t001 Values are means 6 SE; n = 5. P,0.05 Significantly different between NaCl 2 M and NaCl 0.15 M groups.+P,0.05 Significantly different from baseline levels (time 0). Experiment 3: Brain Pattern of Fos, Fos-OT and Fos-5HT Immunoreactivity after SO doi:10 1371/journal pone 0074689 t001 September 2013 \| Volume 8 \| Issue 9 \| e74689 September 2013 \| Volume 8 \| Issue 9 \| e74689 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 5 Control of Sodium Balance by Serotonergic Neurons 1:10,000 in PB containing 2% NHS (Gibco, Auckland, NZ) and 0.3% Triton X-100 (Sigma Chemical Co., St. Louis, MO, USA). The sections were then washed with PB and incubated with biotin- labeled anti-rabbit immunoglobulin and avidin-biotin-peroxidase complex (Vector Laboratories Inc., Burlingame, CA USA, 1:200 dilutions in 1% NHS-PB) for 1 h at room temperature. The peroxidase label was detected using diaminobenzidine hydrochlo- ride (DAB, Sigma Chemical Co., St. Louis, MO, USA), intensified with 1% cobalt chloride and 1% nickel ammonium sulphate. This method produces a blue-black nuclear reaction product. The series of Fos-labeled sections, also processed for immunocytochemical localization of 5HT and OT, were incubated for 72 h at 4uC with their corresponding antibodies: polyclonal rabbit anti-5HT antibody (ImmunoStar Inc, WI, USA, dilution 1:10,000) and polyclonal rabbit anti-OT antibody (Calbiochem, dilution: 1:25,000). After incubation, the sections were rinsed and incubated with biotin-labeled anti-rabbit immunoglobulin and avidin-biotin- peroxidase complex for 1 h at room temperature. Cytoplasmic 5HT-ir and OT-ir were detected with unintensified DAB that produces a brown reaction product. Finally, the free-floating sections were mounted on gelatinized slides, air-dried overnight, dehydrated, cleared in xylene and placed under a coverslip with DePeX (Fluka, Buchs, Switzerland). Table 2. Renal response after 2 M and 0.15 M NaCl infusion. Unit 0.15 M NaCl 2 M NaCl Urinary volume ml. N 100 g bw21 ?3 h 0.533 (60.128) 3.105 (60.255)* Na+ Excretion meq. N 100 g bw21 0.047 (60.015) 0.699 (60.057)* Cl2 Excretion meq. N 100 g bw21 0.446 (60.016) 0.798 (60.071)* K+ Excretion meq. N 100 g bw21 0.038 (60.015) 0.143 (60.024)* Osmolality mosmol. N bw21 692.579 (665.895) 693.826 (627.407) n = 6 n = 9 Values are means 6 SE; n = 5. P,0.05 Significantly different between groups. doi:10.1371/journal.pone.0074689.t002 distribution of sodium overload-induced c-fos in these animals with that observed and reported previously after induced-sodium consumption [9,11,29]. The brains were removed, fixed in the same solution overnight, and then stored at 4uC in PB containing 30% sucrose. Coronal sections were cut into two series of 40 mm using a freezing microtome and were placed in a mixture of 10% H2O2 and 10% methanol until oxygen bubbles ceased appearing. Experiment 3: Brain Pattern of Fos, Fos-OT and Fos-5HT Immunoreactivity after SO They were incubated in 10% normal horse serum (NHS) in PB for 1 h to block non-specific binding sites. Fos-ir controls were conducted by placing sections in primary Fos antibody that had been preadsorbed with an excess of Fos peptide, or by processing sections without the primary antiserum. No Fos-ir neurons were observed following either of these control procedures. All the series of the free-floating sections from each brain were first processed for Fos immunoreactivity (Fos-ir), using an avidin- biotin-peroxidase procedure. The sections of the midbrain were then also stained for 5HT immunoreactivity (5HT-ir) and those from the hypothalamus for OT (OT-ir). The staining procedures followed the double-labeling procedures previously described [29,9]. Briefly, free-floating sections were incubated overnight at room temperature in a rabbit anti-fos antibody (produced in rabbits against a synthetic 14-amino acid sequence, corresponding to residues 4–17 of human Fos) (Ab-5, Calbiochem), diluted Cytoarchitectural and Quantitative Analysis Brain nuclei exhibiting Fos-ir were identified and delimited according to the rat brain atlas of Paxinos and Watson [21]. The different PVN subnuclei were counted at three different levels of PVN, anterior, medial, posterior (distance from the bregma of the corresponding plates: 21.30 mm, 21.80 mm, 22.12 mm respec- Figure 5. Plasma OT concentration before and 5, 10 and 15 minutes after 2 M NaCl or 0.15 M NaCl infusion. Values are means 6 SE. The 2 way ANOVA indicated that the main treatment factor was statistically significant (F1, 45 = 8.3; p = 0.006) P,0.05 difference from 0.15 M NaCl group. doi:10.1371/journal.pone.0074689.g005 Figure 5. Plasma OT concentration before and 5, 10 and 15 minutes after 2 M NaCl or 0.15 M NaCl infusion. Values are means 6 SE. The 2 way ANOVA indicated that the main treatment factor was statistically significant (F1, 45 = 8.3; p = 0.006) P,0.05 difference from 0.15 M NaCl group. doi:10.1371/journal.pone.0074689.g005 doi:10.1371/journal.pone.0074689.g005 September 2013 \| Volume 8 \| Issue 9 \| e74689 September 2013 \| Volume 8 \| Issue 9 \| e74689 PLOS ONE \| www.plosone.org 6 Control of Sodium Balance by Serotonergic Neurons Figure 6. Brain Pattern of Fos-ir and Double–immunolabeled cells, Fos-5HT, after SO. A)- Average number of neurons single-labeled for 5HT, B)- Fos and C)-double-labeled for Fos and 5HT, along the median raphe nucleus and dorsal, ventrolateral and ventral subdivisions of DRN, after s.c. injections of 2 M NaCl or 0.15 M NaCl. Values are means 6 SE. P,0.05 significant differences between sodium overload (2 M NaCl) vs control (0.15 M NaCl) groups. D)- Schematic photomicrographs of DRN and MnR analyzed sections (left panel), illustrating the effects of s.c. injections of 2 M NaCl or 0.15 M NaCl on c-fos expression in serotonergic neurons of DRN (upper and bottom right panels, respectively). Small square within DRD indicates the region photographed at higher magnification (40X), and shown in right panels. Fos-5HT immunolabeled cells within the DRD are indicated by arrows (upper right panel). Scale Bar: 50 mm. doi:10.1371/journal.pone.0074689.g006 Figure 6. Brain Pattern of Fos-ir and Double–immunolabeled cells, Fos-5HT, after SO. A)- Average number of neurons single-labeled for 5HT, B)- Fos and C)-double-labeled for Fos and 5HT, along the median raphe nucleus and dorsal, ventrolateral and ventral subdivisions of DRN, after s.c. injections of 2 M NaCl or 0.15 M NaCl. Values are means 6 SE. Cytoarchitectural and Quantitative Analysis P,0.05 significant differences between sodium overload (2 M NaCl) vs control (0.15 M NaCl) groups. D)- Schematic photomicrographs of DRN and MnR analyzed sections (left panel), illustrating the effects of s.c. injections of 2 M NaCl or 0.15 M NaCl on c-fos expression in serotonergic neurons of DRN (upper and bottom right panels, respectively). Small square within DRD indicates the region photographed at higher magnification (40X), and shown in right panels. Fos-5HT immunolabeled cells within the DRD are indicated by arrows (upper right panel). Scale Bar: 50 mm. doi:10.1371/journal.pone.0074689.g006 Photoshop Image Analysis Program, version 5.5. The counting was done in four animals of each condition, and was repeated at least twice on each section analyzed, to ensure that the number of profiles obtained was similar. The investigator who conducted the counting of Fos-ir cells was blinded for the experimental groups. Immunohistochemical study was analyzed by Student t-test. tively). The distance from the bregma of the corresponding plates is indicated between brackets: accumbens core (AcbC, 1.00 mm), SON (21.3 mm), central amygdaloid nucleus (CeA, 22.3 mm), bed nucleus of the stria terminalis, laterodorsal subdivision (BSTLD, 20.26 mm), subfornical organ (SFO, 20.92 mm), organum vasculosum of the lamina terminalis (OVLT, 20.20 mm), median preoptic nucleus (MnPO, 20.30 mm), thalamic anterodorsal (AD, 21.8 mm), median raphe nucleus (MnR) and DRN (28.00 mm), LPBN (29.3 mm), NTS (213.24 mm) and AP (213.68 mm). tively). The distance from the bregma of the corresponding plates is indicated between brackets: accumbens core (AcbC, 1.00 mm), SON (21.3 mm), central amygdaloid nucleus (CeA, 22.3 mm), bed nucleus of the stria terminalis, laterodorsal subdivision (BSTLD, 20.26 mm), subfornical organ (SFO, 20.92 mm), organum vasculosum of the lamina terminalis (OVLT, 20.20 mm), median preoptic nucleus (MnPO, 20.30 mm), thalamic anterodorsal (AD, 21.8 mm), median raphe nucleus (MnR) and DRN (28.00 mm), LPBN (29.3 mm), NTS (213.24 mm) and AP (213.68 mm). Experiment 1: Effects of a Body Sodium Overload on Putative 5HT-DRN Neuronal Firing Fos-ir nuclei were quantified using a computerized system that includes a Zeiss microscope equipped with a DC 200 Leica digital camera attached to a contrast enhancement device. Images were digitalized and analyzed using Scion Image PC, based on the NIH 1997 version. Fos-ir cells in each section were counted by setting a size range for cellular nuclei (in pixels) and a threshold level for staining intensity. Representative sections in each group were acquired at exactly the same level, with the aid of the Adobe The effect of a 2 M NaCl infusion was tested in 20 putative serotonergic neurons of the DRN. Of the 20 neurons tested with s.c. NaCl 2 M infusion, 14 (70%) displayed an excitatory response, 3 (15%) an inhibitory response and 3 (15%) did not respond. Of the 16 neurons infused with isotonic saline, 3 (19%) displayed an excitatory response, 6 (37.5%) an inhibitory response and 7 (43.5%) did not respond. Taking into account this heterogeneity of September 2013 \| Volume 8 \| Issue 9 \| e74689 7 PLOS ONE \| www.plosone.org Control of Sodium Balance by Serotonergic Neurons Figure 7. Brain Pattern of Fos-ir and Double–immunolabeled cells, Fos-OT, after SO. A)- Average number of Fos immunoreactive neurons and B)- Fos-OT immunolabeled neurons in the supraoptic nucleus (SON), and paraventricular hypothalamic nucleus along the lateral magnocellular and medial subdivisions (PaLM and PaMM, respectively), after s.c. injections of 2 M NaCl or 0.15 M NaCl. Values are means 6 SE. P,0.05 significant differences between overload (2 M NaCl) vs. control (0.15 M NaCl) groups. The bottom panels photomicrographs (C,D,E) are showing the pattern of double Fos-OT immunoreactive cells in the SON, C)-, and paraventricular hypothalamic nucleus along the medial (PaMM, D) and lateral magnocellular (PaLM, E) subdivisions in control (upper sections) and sodium overload animals (bottom sections). The left panels illustrate the distribution of these immunoreactive cells at low magnification (10x). Small squares in these panels indicate regions photographed at higher magnification (40x, right panels), and indicated by arrows are the cells photographed at higher magnification (1006). Scale Bar: 100 mm. doi:10.1371/journal.pone.0074689.g007 Figure 7. Brain Pattern of Fos-ir and Double–immunolabeled cells, Fos-OT, after SO. A)- Average number of Fos immunoreactive neurons and B)- Fos-OT immunolabeled neurons in the supraoptic nucleus (SON), and paraventricular hypothalamic nucleus along the lateral magnocellular and medial subdivisions (PaLM and PaMM, respectively), after s.c. injections of 2 M NaCl or 0.15 M NaCl. Experiment 2: Physiological Characterization of the Response to Sodium Overload Water intake induced by SO. As previously described, water intake significantly increased in the 2 M NaCl injected group compared to the 0.15 M NaCl group (Fig. 4). That is, the interaction between treatment factor and time was statistically significant (F4,64 = 4.059; p = 0.005), increasing the volume of water drunk by 2 M NaCl rats. Sodium overload rats started to drink water 31 minutes after 2 M NaCl infusion (means of 9 rats). Most of the control rats did not drink water during the test of 180 minutes (6 rats), and those who drank water started to drink 82 minutes after 0.15 M NaCl injection (means of 3 animals). Figure 3 shows the representative plots at a rostral (3.A) and caudal level (3.B) of DRN from subjects with sodium or control overload, to illustrate the precise location of the 14 recording sites where the dye injection was successfully delivered, specifying the different responses: excitatory (+), inhibitory(2), control () and neutral (O) responses. In the remaining cases we have only an estimation of the recording site location; however all were approximately sited within the dorsal, ventral and ventrolateral Experiment 1: Effects of a Body Sodium Overload on Putative 5HT-DRN Neuronal Firing Values are means 6 SE. P,0.05 significant differences between overload (2 M NaCl) vs. control (0.15 M NaCl) groups. The bottom panels photomicrographs (C,D,E) are showing the pattern of double Fos-OT immunoreactive cells in the SON, C)-, and paraventricular hypothalamic nucleus along the medial (PaMM, D) and lateral magnocellular (PaLM, E) subdivisions in control (upper sections) and sodium overload animals (bottom sections). The left panels illustrate the distribution of these immunoreactive cells at low magnification (10x). Small squares in these panels indicate regions photographed at higher magnification (40x, right panels), and indicated by arrows are the cells photographed at higher magnification (1006). Scale Bar: 100 mm. doi:10.1371/journal.pone.0074689.g007 subdivisions of the DRN between 27.8 mm and 28.0 mm from bregma. subdivisions of the DRN between 27.8 mm and 28.0 mm from bregma. responses, the quantitative analysis was done comparing the excitatory putative 5HT neurons of the sodium overload vs control group. The analysis showed a significant interaction between the treatment and time (F(18,504) =5.82; P = 0.001). That is, there is a significant differential response in excitatory neuronal firing of the 5HT cells of rats infused with a hypertonic solution of sodium (2 M NaCl) compared to those infused with isotonic saline (0.15 M NaCl) (Figs. 1 and 2). Post hoc analysis indicated that the firing rate of putative serotonergic neurons in rats infused with 2 M NaCl increased significantly 3 and 4 minutes after the infusion started compared to the 0.15 M NaCl injected group and to baseline, respectively. In contrast, the treatment with isotonic saline induced a tendency to decrease the firing rate of serotonergic neurons, being significantly different in the last point of time. Besides, in those neurons that showed an inhibitory response induced by 2 M NaCl, inhibition was observed 4 minutes after the infusion began compared to baseline level. Experiment 3: Brain Pattern of Fos-ir and Double– immunolabeled Cells (Fos-5HT and Fos-OT) after s.c. Infusion of 2 M NaCl vs 0.15 M NaCl Solutions DRN. As observed in our previous studies, greater activation was found at medial level of the DRN, which includes its dorsal, ventral and ventrolateral subdivisions. As expected based on previous results, sodium overload produced a significant increase in the number of Fos immunoreactive neurons in the DRD, DRV, and DRVL subdivisions of DRN. The number of double-labelled (Fos-5HT) cells is also increased in the DRD and DRVL regions of DRN (Fig. 6). No significant differences were observed in the Fos- ir and Fos-5HT neurons in the other raphe nuclei such as MnR (Figs. 6 B and C respectively). We also analyzed the number of 5HT neurons in both groups; however we did not observe any significant differences in these analyzed areas (Fig. 6A). DRN. As observed in our previous studies, greater activation was found at medial level of the DRN, which includes its dorsal, ventral and ventrolateral subdivisions. As expected based on previous results, sodium overload produced a significant increase in the number of Fos immunoreactive neurons in the DRD, DRV, and DRVL subdivisions of DRN. The number of double-labelled (Fos-5HT) cells is also increased in the DRD and DRVL regions of DRN (Fig. 6). No significant differences were observed in the Fos- ir and Fos-5HT neurons in the other raphe nuclei such as MnR (Figs. 6 B and C respectively). We also analyzed the number of 5HT neurons in both groups; however we did not observe any significant differences in these analyzed areas (Fig. 6A). The analysis of plasma osmolality also showed a significant interaction between treatment factor and time (F3, 64 = 3.95; p = 0.012). As shown in Table 1, an increase was observed in the sodium overload group at 5, 10, 15 minutes after 2 M NaCl s.c. infusion compared to the 0.15 M NaCl group, and at 10 and 15 minutes compared to baseline levels. The percentage increase of plasma osmolality was 3–4% compared to baseline levels. Renal response induced by sodium overload. Sodium overloaded rats had a different renal response compared to the control group during the 180 minutes after s.c. injection (Table 2). Three of the nine rats injected with isotonic saline did not even urinate, while all the rats infused with hypertonic NaCl showed this response. As shown in Table 2, a significant increase in sodium, chloride and potassium renal excretion was observed after 2 M NaCl s.c. Experiment 3: Brain Pattern of Fos-ir and Double– immunolabeled Cells (Fos-5HT and Fos-OT) after s.c. Infusion of 2 M NaCl vs 0.15 M NaCl Solutions infusion compared to the control infused group. However, no difference was found between groups in urine osmolality. SON and PVN. Fos expression in the SON was significantly increased in sodium overloaded animals compared to the control group. As previously shown [5,4], the number of double- immunolabeled neurons for Fos and oxytocin was also significantly increased in the 2 M NaCl group (Fig. 7). Within the medial (PaMM) and lateral (PaLM) magnocellular subdivisions of the PVN, a significant increase in Fos and Fos-OT immunoreactive cells was found, 90 min after s.c. 2 M NaCl infusion (Fig. 7). Plasma Protein Concentration. Osmolality and Plasma Sodium and Urinary Concentration As expected, 2 M NaCl infusions significantly increased plasma sodium concentra- tion and osmolality. The ANOVA for plasma sodium concentra- tion indicated a significant interaction between treatment factor and time (F3, 64 = 4.3; p = 0.008). Plasma sodium concentration significantly increased at 5, 10, 15 minutes compared to the control group and also increased at 10 minutes compared to baseline levels (Table 1). Experiment 3: Brain Pattern of Fos-ir and Double– immunolabeled Cells (Fos-5HT and Fos-OT) after s.c. Infusion of 2 M NaCl vs 0.15 M NaCl Solutions Plasma Protein Concentration. Osmolality and Plasma Sodium and Urinary Concentration In order to infer whether there are blood volume changes after s.c. saline infusions, plasma protein concentration was examined. No significant differences were observed in plasma protein concentration for treatment factor or time, suggesting that there are no changes in blood volume between groups or baseline levels (Table 1). September 2013 \| Volume 8 \| Issue 9 \| e74689 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 8 Control of Sodium Balance by Serotonergic Neurons Figure 8. Brain Pattern of Fos-ir in the brainstem nuclei after SO. A)- Average number of Fos-immunoreactive neurons in the NTS, AP and LPBN after s.c. injections of 2 M NaCl or 0.15 M NaCl. Values are means 6 SE. P,0.05 significantly different from 0.15 M NaCl group. B)- Photomicrographs showing the pattern of Fos-immunoreactivity within the LPBN (A–B), AP (C–D) and NTS (E–F), after s.c. injections of 2 M NaCl (B,D,F) or 0.15 M NaCl (A, C, E). Scale Bar: 100 mm. doi:10.1371/journal.pone.0074689.g008 Figure 8. Brain Pattern of Fos-ir in the brainstem nuclei after SO. A)- Average number of Fos-immunoreactive neurons in the NTS, AP and LPBN after s.c. injections of 2 M NaCl or 0.15 M NaCl. Values are means 6 SE. P,0.05 significantly different from 0.15 M NaCl group. B)- Photomicrographs showing the pattern of Fos-immunoreactivity within the LPBN (A–B), AP (C–D) and NTS (E–F), after s.c. injections of 2 M NaCl (B,D,F) or 0.15 M NaCl (A, C, E). Scale Bar: 100 mm. doi:10.1371/journal.pone.0074689.g008 Plasma osmolality and electrolytes. As expected, 2 M NaCl infusions significantly increased plasma sodium concentra- tion and osmolality. The ANOVA for plasma sodium concentra- tion indicated a significant interaction between treatment factor and time (F3, 64 = 4.3; p = 0.008). Plasma sodium concentration significantly increased at 5, 10, 15 minutes compared to the control group and also increased at 10 minutes compared to baseline levels (Table 1). The analysis of plasma osmolality also showed a significant interaction between treatment factor and time (F3, 64 = 3.95; p = 0.012). As shown in Table 1, an increase was observed in the sodium overload group at 5, 10, 15 minutes after 2 M NaCl s.c. infusion compared to the 0.15 M NaCl group, and at 10 and 15 minutes compared to baseline levels. The percentage increase of plasma osmolality was 3–4% compared to baseline levels. Plasma osmolality and electrolytes. Control of Sodium Balance by Serotonergic Neurons Control of Sodium Balance by Serotonergic Neurons The endocrine, renal and behavioral responses were also analyzed in the same model and the brain areas and the oxytocinergic PVN-SON groups of neurons activated after a sodium overload were identified (by single and double immuno- labeling). Lamina terminalis. The 2 M NaCl group showed a significant increase in the number of Fos-ir neurons along the circumventricular organs of the lamina terminalis, OVLT and SFO, compared to the control group. A significant increase in the number of Fos-ir cells of experimental group was also observed in the ventral part of the MnPO (Fig. 9). Our data provide new evidence regarding the activation of specific groups of serotonergic cells within the DRN during increases of plasma sodium concentration and osmolarity not associated to volume expansion. Central Extended Amygdala (ExA). A significantly in- creased number of activated neurons were observed within the central extended amygdala nuclei, CeA and BSTLD of sodium- overloaded rats, in comparison with control rats (Fig. 10). The electrophysiological evidence supports previous immuno- histochemical and pharmacological results that involved seroto- nergic pathways modulating sodium intake and renal excretion after different hydroelectrolytic disturbances. Our previous reports demonstrated serotonergic system involvement in the inhibitory control of sodium appetite induced by peritoneal dialysis (PD). Fos-ir decreased in serotonergic cells 24 hs after PD (during the appetitive phase) compared to non-depleted animals, while Fos-ir increased during the satiety phase when animals were in process of reestablishing body sodium status by ingesting sodium salts [9,11,12]. According to these, it has been shown that fos expression depends on the temporal features of action potential patterns. For example in cultured dorsal root ganglion cells, immediate-early gene activation was inversely correlated with the burst –intervals of action potentials [30]. Our previous results also demonstrate that the 5HT system is involved in the regulation of renal responses. Serotonergic receptor blockade with LPBN injections of methysergide reduced the increase in urinary volume and sodium and potassium excretion induced by EVE, while injections of the serotonergic 5HT2A and 5HT2C receptor agonist, 2.5-dimethoxy-4-iodoamphetamine hydrobromide, en- hanced the effects of BVE on Na_ and K_ excretion and urinary volume [20]. Decreased levels of 5HT and its metabolite 5- Brain Pattern of Fos-ir Neurons in other Nuclei not Involved in Sodium Balance Regulation In order to demonstrate that the sodium-overload induced effect is selective to brain regions of interest, and not a global elevation in neuronal activity throughout the brain we included the analysis of others nuclei such as AD and AcbC. In both areas we did not observed significantly differences between sodium overload and control groups (AD: mean of 2 M NaCl: 15.17, mean of 0.15 M NaCl: 15.4, p = 0.97 and AcbC: mean of 2 M NaCl: 20.8, mean of 0.15 M NaCl: 22, p = 0.91). Plasma OT Concentration As previously demonstrated [5,4], hypertonic sodium infusion changed plasma OT concentration compared to the control group.The ANOVA indicated that the main treatment factor was statistically significant (F1, 45 = 8.305; p = 0.006), but the interac- tion between the treatment factor and time did not reach significant levels (Fig. 5). Brainstem: The t-test analysis indicated that sodium overload produces a significant increase in the number of Fos immunore- active cells along the AP, NTS and LPBN compared to control animals (Fig. 8). September 2013 \| Volume 8 \| Issue 9 \| e74689 PLOS ONE \| www.plosone.org 9 Control of Sodium Balance by Serotonergic Neurons Discussion B)- Photomicrographs showing the pattern of Fos-immunoreactivity within the CeA (A–B) and BSTLD (C–D), after s.c. injections of 2 M NaCl (B,D,F) or 0.15 M NaCl (A, C, E). Scale Bar: 100 mm. doi:10.1371/journal.pone.0074689.g010 hydroxyindoleacetic acid were also observed within the raphe nucleus 15 min after EVE [14]. Finally, the activation and the increase in serotonergic DRN firing frequency after a body sodium overload may be interpreted as these cells participate in the behavioral osmoregulatory response [9–11]. It would also influ- ence renal and endocrine responses, increasing plasma OT and atrial natriuretic peptide and consequently increasing urine output and sodium and potassium renal excretion, as often reported after an EVE or after induced sodium intake [10,11,20]. observed that the SO group consumed three times more water than the control group during the 3 h drinking test. Renal response is also implicated in plasma tonicity regulation. In agreement with previous reports, we found that SO enhanced volume and electrolyte excretion [39,40]. High sodium excretion is in part a consequence of the natriuretic effect of plasma oxytocin, which was also found to be elevated. These data confirm previous studies which showed that OT is released after hyperosmotic stimulus [41–43]. The observed variations in osmolarity and plasma sodium concentration produced by subcutaneous sodium overload are detected by highly specialized neurons, able to translate these changes to electrical signals which activate CNS areas involved in the control of water and salt intake and excretion [44–46]. There is a general agreement that osmosensory transduction is primarily mediated by cells of the two circumventricular organs located in the lamina terminalis: the SFO and the OVLT [44,45,47–50]. These areas send information to the DRN [13] and hypothalamic nuclei such as PVN, SON and MnPO [50–52]. Then they may activate the magnocellular neurons of the PVN and SON, increasing the plasma concentration of OT. As previously described in the results section, sodium overload also increased Fos-ir and Fos-OT positive cells in the SON after 2 M NaCl infusion; however, isotonic infusion did not produce any change in oxytocinergic cell activity. These data together suggest that the serotonergic system at DRN level is modulated by body sodium status and is therefore participating in its regulation. Discussion The results of this study allow us to determine in an ‘‘in vivo" model that DRN 5HT neurons increase their firing frequency during an increase in systemic sodium concentration and osmolality, matching the pattern of Fos-5HT double immuno- staining. Therefore, both electrical activity (analyzed by in vivo extracellular recording) and c-fos expression within serotonergic cells of the DRN increased after a hypernatremic/hyperosmolar state. Figure 9. Brain Pattern of Fos-ir in the Lamina Terminalis nuclei after SO. A)- Average number of Fos-immunoreactive neurons in the SFO, OVLT and MnPO after s.c. injections of 2 M NaCl or 0.15 M NaCl. Values are means 6 SE. P,0.05 significantly different from 0.15 M NaCl group. B)- Photomicrographs showing the pattern of Fos-immunoreactivity within the SFO (A–B), OVLT (C–D) and MnPO (E–F), after s.c. injections of 2 M NaCl (B,D,F) or 0.15 M NaCl (A, C, E). Scale Bar: 100 mm. doi:10.1371/journal.pone.0074689.g009 Figure 9. Brain Pattern of Fos-ir in the Lamina Terminalis nuclei after SO. A)- Average number of Fos-immunoreactive neurons in the SFO, OVLT and MnPO after s.c. injections of 2 M NaCl or 0.15 M NaCl. Values are means 6 SE. P,0.05 significantly different from 0.15 M NaCl group. B)- Photomicrographs showing the pattern of Fos-immunoreactivity within the SFO (A–B), OVLT (C–D) and MnPO (E–F), after s.c. injections of 2 M NaCl (B,D,F) or 0.15 M NaCl (A, C, E). Scale Bar: 100 mm. doi:10.1371/journal.pone.0074689.g009 September 2013 \| Volume 8 \| Issue 9 \| e74689 PLOS ONE \| www.plosone.org 10 Control of Sodium Balance by Serotonergic Neurons Figure 10. Brain Pattern of Fos-ir in the Central Extended Amygdala nuclei after SO. A)- Average number of Fos-ir neurons in the CeA and BSTLD after s.c. injections of 2 M NaCl or 0.15 M NaCl. Values are means 6 SE. P,0.05 significantly different from 0.15 M NaCl group. B)- Photomicrographs showing the pattern of Fos-immunoreactivity within the CeA (A–B) and BSTLD (C–D), after s.c. injections of 2 M NaCl (B,D,F) or 0.15 M NaCl (A, C, E). Scale Bar: 100 mm. doi:10.1371/journal.pone.0074689.g010 Figure 10. Brain Pattern of Fos-ir in the Central Extended Amygdala nuclei after SO. A)- Average number of Fos-ir neurons in the CeA and BSTLD after s.c. injections of 2 M NaCl or 0.15 M NaCl. Values are means 6 SE. *P,0.05 significantly different from 0.15 M NaCl group. September 2013 \| Volume 8 \| Issue 9 \| e74689 Discussion In sum, the increased activity and firing frequency of putative serotonergic cells of the DRN after body sodium overload may reflect how this system mediates the behavioral, renal and endocrine responses for reestablishing body sodium balance. Neuroanatomical evidence indicates that the DRN sends 5HT projections to the LPBN [14,31], forming a key pathway to regulate homeostatic responses under hydroelectrolyte balance alterations. Previous investigations also demonstrated that seroto- nergic mechanisms in the LPBN play an inhibitory role in controlling sodium appetite following a variety of dipsogenic and/ or natriorexigenic stimuli [17,32–35]. Both the LPBN and the DRN receive afferents from the LT nuclei and their cells are activated not only after SO but also during different body sodium balance changes [13,12]. That is, hypertonicity would be detected by the LT, which sends projections directly to the DRN and LPBN, increasing the activity of DRN 5HT cells, which also send efferents to the LPBN, modulating its activity and consequently sodium appetite. Different areas of the brainstem such as the NTS, AP, LPBN, which mediate peripheral satiety and osmoregulatory signals to modulate fluid intake and neurohypophyseal hormone secretion, showed increased Fos immunoreactivity after SO, and structures of the extended amygdala complex, such as the CeA and bed nucleus of the stria terminalis (BST), which are involved in the processing of integrated signals related to sodium appetite behavior, are activated by SO [4–8,53,54]. These data give new evidence for BST involvement during systemic hypertonicity states associated with normo-volemia. Previous studies indicated that both the CeA and BST sub-nuclei are also activated by isotonic and hypertonic EVE and are involved in thirst and sodium appetite control [10,55–57]. As expected, SO significantly increased plasma sodium concentration and osmolality. The highest increase was observed at 10 minutes of NaCl injection. However, no differences in plasma protein concentration were observed, suggesting that this protocol of SO does not involve an EVE. As previously described [36–39], an increase of 1–2% in plasma osmolality produces the activation of a central circuit that induces thirst in order to reestablish normal osmolality. Similarly, we September 2013 \| Volume 8 \| Issue 9 \| e74689 PLOS ONE \| www.plosone.org 11 Control of Sodium Balance by Serotonergic Neurons body sodium status, inhibiting sodium appetite and increasing natriuresis. 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Godino A, Margatho LO, Caeiro XE, Antunes-Rodrigues J, Vivas L (2010) Activation of lateral parabrachial afferent pathways and endocrine responses during sodium appetite regulation. Exp Neurol.;221(2): 275–84. 34. Davern PJ, McKinley MJ (2010) Forebrain regions affected by lateral parabrachial nucleus serotonergic mechanisms that influence sodium appetite. Brain Res 1339, 41–48. 13. Badaueˆ-Passos D Jr, Godino A, Johnson AK, Vivas L, Antunes-Rodrigues J (2007) Dorsal raphe nuclei integrate allostatic information evoked by depletion induced sodium ingestion. Exp Neurol. 206: 86–94. 35. Acknowledgments The authors thank Graciela Jahn and Marta Soaje for their help on measuring oxytocin plasma concentration. Discussion In conclusion, taking into account these results and other studies, we can speculate on the possible brain circuit involved in regulatory responses during a SO without volume expansion. Lamina terminalis structures detect humoral changes produced by SO such as plasma hormones, Na concentration and osmolality changes. The circumventricular organs of the LT send projections to the hypothalamus (SON and PVN), brainstem nuclei (LPBN, NTS, AP and DRN) and central extended amygdala complex. LT projections, among others, activate the oxytocinergic neurons of SON and PVN, increasing plasma OT concentration in order to promote sodium excretion. SO produces an increase in Fos-ir and electrical activity of 5HT DRN neurons. These cells may control body sodium status, inhibiting sodium appetite and increasing natriuresis. Author Contributions Conceived and designed the experiments: AG HFC LV. Performed the experiments: AG SP. Analyzed the data: AG SP HFC. Contributed reagents/materials/analysis tools: AG HFC LV. Wrote the paper: AG SP HFC LV. Technical assistance of experiments electrophysiology: HC. Control of Sodium Balance by Serotonergic Neurons 43. 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(1995) Oxytocin mediates atrial natriuretic peptide release and natriuresis after volume expansion in the rat. Proc Natl Acad Sci U S. A 92: 7902–7906. PLOS ONE \| www.plosone.org September 2013 \| Volume 8 \| Issue 9 \| e74689 12 September 2013 \| Volume 8 \| Issue 9 \| e74689 September 2013 \| Volume 8 \| Issue 9 \| e74689 Control of Sodium Balance by Serotonergic Neurons McKinley MJ, Hards DK, Oldfield BJ (1994) Identification of neural pathways activated in dehydrated rats by means of Fos-immunohistochemistry and neural tracing. Brain Res. 8;653(1–2): 305–14. September 2013 \| Volume 8 \| Issue 9 \| e74689 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 13
https://openalex.org/W3113175035	https://www.e3s-conferences.org/articles/e3sconf/pdf/2020/78/e3sconf_iseese2020_04006.pdf	English	null	Research on the Problems and Development of Small and Medium Enterprises in the Pearl River Delta	E3S web of conferences	2,020	cc-by	2,914	Research on the Problems and Development of Small and Medium Enterprises in the Pearl River Delta Liang Huang1,a, Haowei Ti1,a, Leigang Pei1,a, Ding Ma2,b 1Hong Kong Asia Business College Headquarter Office, Hong Kong 2Limkokwing University of Creative Technology China Office Zhengzhou, China Liang Huang1,a, Haowei Ti1,a, Leigang Pei1,a, Ding Ma2,b 1Hong Kong Asia Business College Headquarter Office, Hong Kong 2Limkokwing University of Creative Technology China Office Zhengzhou, China Abstract—Since the reform and opening up, the economy of the Pearl River Delta has achieved rapid development and remarkable results. Small and medium-sized enterprises in the region have also achieved rapid development in terms of quantity and quality, and have become an important part of the region. However, with economic globalization, because of continuous changes in the external environment, and its own industrial upgrading and structural adjustment, even if small and medium-sized enterprises in the Pearl River Delta region have advantages compared with other regions in China. However, there are still problems in the development process. The article mainly analyzes the financing difficulties of SMEs in the Pearl River Delta region and the brain drain in recent years, and gives corresponding countermeasures. Since small and medium-sized enterprises occupy an important position in the economy of the Pearl River Delta region, they can not only promote technological innovation and boost domestic demand for production, but also drive the employment rate to rise. Therefore, the development of small and medium-sized enterprises in the region has played a positive role in the economic progress of the Pearl River Delta region, and the place has also promoted economic development. conditions for most SMEs to expand their production scales. E3S Web of Conferences 218, 04006 (2020) ISEESE 2020 E3S Web of Conferences 218, 04006 (2020) ISEESE 2020 https://doi.org/10.1051/e3sconf/202021804006 1 INTRODUCTION Due to the limitations of their own scale and strength, in order to achieve the purpose of saving costs, some small and medium-sized enterprises lack professional finance, so there will be irregular financial statements. Data shows that about 60% of SMEs cannot provide long-term standardized financial statements. Moreover, these companies will use inconsistent accounts to reduce taxes or reduce costs, which reduces the cost of SMEs. But as a result, the trust between banks and enterprises has declined, reducing the willingness of financial institutions to lend. For most SMEs, it is not easy to obtain loans provided by financial institutions. First, these organization have higher requirements for high-quality collateral. Secondly, SMEs are not competitive in the quantity and quality of collateral required by financial institutions due to their own scale limitations and strength. What’s more, the real interest rate of SMEs in the loan process is much higher than the benchmark interest rate. Data from the Guangdong Provincial Government in 2017 showed that the actual loan interest rate provided by financial institutions to SMEs was 18.4% higher than the benchmark interest rate on average. The business model and the form of private operation are the main characteristics of SMEs in the Pearl River Delta. However, most of the private companies are small in scale and not standardized. Moreover, many enterprises are developed from small family workshops, and the quality and capabilities of managers is not high. The financing channels are mainly divided into internal financing and external financing. External financing has an incomparable advantage over internal financing. Since external financing is mainly commercial bank loans, which is not only more stable, but also cheaper than private loans. However, commercial banks have relatively high credit requirements for corporate loans, which makes it difficult for most SMEs to meet this type of loan. The endogenous financing preferred by most SMEs refers to the use of internal funds to maintain the operation of the enterprise. The amount of money raised in this way is limited, and in most cases cannot meet the © 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/). aedu.china@139.com, b15303964@qq.com 2.1 The not perfect enterprise management system and unreasonable structure 2.1 The not perfect enterprise management system and unreasonable structure aedu.china@139.com, b15303964@qq.com E3S Web of Conferences 218, 04006 (2020) ISEESE 2020 https://doi.org/10.1051/e3sconf/202021804006 Figure 1 Turnover rate in different industries in Guangdong province 31% 24% 17% 28% Lighting Manufacturing Home appliance manufacturing Packaging and Printing Industry Catering Figure 1 Turnover rate in different industries in Guangdong province employees in the long-term interaction process, and it must be linked to the business performance of the company to help the growth of the company. Many small and medium-sized enterprises in the Pearl River Delta region do not pay attention to the construction of their own corporate culture, and they lack the "people-oriented" corporate temperament. This situation can lead to insufficient corporate cohesion and employee centripetal force, and a weak sense of belonging to the company. In the internal management of some SMEs, managers are too individualistic, while the professional opinions of SME professionals are not taken seriously. The survey results showed that in 2014, nearly 60% of private enterprises in the Pearl River Delta faced severe job shortages. As can be seen from the above figure, different industries generally face rigorous personnel turnover problems. The survey results showed that in 2014, nearly 60% of private enterprises in the Pearl River Delta faced severe job shortages. As can be seen from the above figure, different industries generally face rigorous personnel turnover problems. Figure 2 The turnover rate of small and medium-sized enterprises in Guangdong province 0% 5% 10% 15% 20% 25% 30% 35% Restricted by the family-style management model, many small and medium-sized enterprises in the Pearl River Delta region hire and promote employees based on their blood relationship, resulting in mismatches in talents, positions and salaries. This kind of unfair competition makes it difficult to form an atmosphere of respect for knowledge and talents in the development of enterprises. In terms of management mode, the distrust, unauthorization or insufficient authorization of enterprise managers and subordinates makes key talents play the role of executor, which is not conducive to the cultivation of enterprise groups. As a result, there is no open and fair competition mode within the enterprise. Figure 2 The turnover rate of small and medium-sized enterprises in Guangdong province With the development of the country’s political economy, the country has the ability to give priority planning and assistance to the regional development. 2.1 The not perfect enterprise management system and unreasonable structure Especially in recent years, with the development of the western region and the construction of a new countryside, job seekers have a wider range of employment options. They are more willing to choose cities and regions in the Pearl River Delta where wages, benefits and living costs are more advantageous. At the same time, the imperfection of the public service functions of local governments hinders job seekers to a certain extent. Figure 2 is mainly a statistical chart drawn from the data officially released by the Guangdong Provincial Department of Labor and Social Security. As we can see that the turnover rate of senior managers and senior technical personnel of SMEs in Guangdong Province is as high as 20% to 30%, and the turnover rate of middle and grassroots managers and technical personnel is 10% to 20%. The normal turnover of 8% is reasonable, which obviously shows that the turnover rate of Chinese private enterprises is abnormally high. 2.2 The lack of advanced corporate culture atmosphere The value of corporate culture refers to the interpersonal communication paradigm and system formed by the 2 https://doi.org/10.1051/e3sconf/202021804006 E3S Web of Conferences 218, 04006 (2020) ISEESE 2020 E3S Web of Conferences 218, 04006 (2020) 3 SUGGESTIONS ON THE DEVELOPMENT OF SMALL AND MEDIUM ENTERPRISES IN THE PEARL RIVER DELTA REGION played a decisive role in the market through the process of macro-control, and has continuously improved the financial system. The multiple links involved in supply chain financial financing require perfect early warning of risks in the financing process. Therefore, the financial institution supervision department should analyze the risk characteristics, credit technology and core value of the supply chain on specific issues, and make a correct assessment. It is necessary to improve the human resource management system of the enterprise, attach importance to the professionalization and systematic training of employees. Incentives can be used to keep employees' hearts. Enterprises must learn to care for employees, and treat them as an indispensable member of the company, not just a resource that can come and go at will. The local government of the Pearl River Delta should improve its service functions, create a good employment environment and social environment, and increase the attraction of the region to foreign job seekers. The government will step up labor supervision and maintain the social employment environment. A sound social security system, medical security for foreign personnel, pension and other security systems can guarantee their lives. Good employment service and talent market, as well as perfect employment platform, can solve the problem of employment and talent shortage in enterprises. At the same time, the company should establish a stable long-term development strategy suitable for the company, and coordinate the corporate salary management, performance management and talent management. What’s more, professional recruitment enterprises carry out extensive cooperation to establish their own recruitment channels and achieve diversified recruitment channels. 3.1 Proposals for small and medium enterprises financing For my country's current small and medium-sized enterprises, it is necessary to vigorously build a talent reserve management system in line with the enterprise, and pay attention to the all-round and comprehensive training of recruited personnel.At the same time, the human resources management department should formulate a practical, reasonable, scientific and feasible talent introduction system. Not only must we not judge heroes based on academic qualifications, but also more in line with the actual work ability and work attitude of talents. Only in this way can the company be invincible in the fierce competition. 3.1 Proposals for small and medium enterprises financing The government should improve the supply chain financial system, strengthen relevant regulations, guidance and supervision under the jurisdiction of the government, form a complete financial policy system for the development of small and medium-sized enterprises. Only sound laws on SMEs can guarantee the direct flow of funds to these companies and promote the healthy development of supply chain finance. At the same time, according to management needs, the way of establishing a credit rating system and standards for small and medium-sized enterprises in the Pearl River Delta region as well as a credit model and database can provide references for the construction of China's social credit collection system and credit intermediary institutions, and effectively collect and evaluate information. The 21st century is the century of talents. At present, the fiercest competition among enterprises is the competition of talents. Talents play an increasingly important role in an enterprise and are the soul engineers of an enterprise.Only by vigorously developing the establishment of a management system for the introduction and training of talents can enterprises achieve long-term sustainable development.A perfect talent reserve system is a key factor in the long-term development and success of an enterprise. For my country's current small and medium-sized enterprises, it is necessary to vigorously build a talent reserve management system in line with the enterprise, and pay attention to the all-round and comprehensive training of recruited personnel.At the same time, the human resources management department should formulate a practical, reasonable, scientific and feasible talent introduction system. Not only must we not judge heroes based on academic qualifications, but also more in line with the actual work ability and work attitude of talents. Only in this way can the company be invincible in the fierce competition. The 21st century is the century of talents. At present, the fiercest competition among enterprises is the competition of talents. Talents play an increasingly important role in an enterprise and are the soul engineers of an enterprise.Only by vigorously developing the establishment of a management system for the introduction and training of talents can enterprises achieve long-term sustainable development.A perfect talent reserve system is a key factor in the long-term development and success of an enterprise. 3.2 Increasing attractiveness of small and medium enterprises  Most SMEs in the Pearl River Delta region have a single business model and single products. However, since the decision-making power of most SMEs is in the hands of a core person, operators can make correct judgments and decisions on market changes in a timely manner, and find gaps in supply chain finance. Core managers can focus on areas neglected by large companies, improve their competitiveness, and gain a firm foothold in the market. Therefore, SMEs can use their various advantages to increase their visibility and credibility with high-quality products, thereby attracting the attention of supply chain financial service providers such as banks. After the introduction of talents, the management department has made a corresponding detailed and thorough training plan for the talents, so that every new employee can become familiar with the working environment and their work content as soon as possible.During the internship period, the most suitable job position is arranged according to the specific performance of each employee according to his own situation. At the same time, the company strictly prohibits the use of nepotism to enter the company's work. In a fair and transparent management environment, every After the introduction of talents, the management department has made a corresponding detailed and thorough training plan for the talents, so that every new employee can become familiar with the working environment and their work content as soon as possible.During the internship period, the most suitable job position is arranged according to the specific performance of each employee according to his own situation. At the same time, the company strictly prohibits the use of nepotism to enter the company's work. In a fair and transparent management environment, every  The development of financial institutions in a market economy is full of opportunities and challenges. In recent years, the government has  The development of financial institutions in a market economy is full of opportunities and challenges. In recent years, the government has 3 3 E3S Web of Conferences 218, 04006 (2020) E3S Web of Conferences 218, 04006 (2020) ISEESE 2020 https://doi.org/10.1051/e3sconf/202021804006 employee will become the backbone of the company.The company adopts long-term quality training combined with short-term job training to effectively combine practice and experience, so that every employee can make a clear position for himself during the internship and bring the greatest benefits to the company. 6. Gan Xiaoli, Li Yan. 3.2 Increasing attractiveness of small and medium enterprises Problems and countermeasures in the development of foreign trade of small and medium-sized enterprises in Guangdong Province[J]. Entrepreneur World Semi-Monthly (Theory Edition), 2009(03): 240-241. 7. Kang Shuyao: Research on Financing Innovation of my country's Small and Medium-sized Enterprises-Taking the Pearl River Delta Region as an Example [J]. Business Accounting, 2018 (20): 73-75. ACKNOWLEDGMENT It took nearly a month to finally finish this paper. I encountered countless difficulties and obstacles in the process of writing the paper. I would like to thank Dr. Haowei, my thesis guide, for his selfless guidance and help. Take the trouble to help modify and improve the paper. I would like to express my most central gratitude to the teachers who have helped and guided me!Thanks to the scholars involved in this paper. This article cites the research literature of several scholars. Without the help and inspiration of the research results of all scholars, it would be difficult for me to finish the writing of this article. Thanks to my colleagues and friends for giving me a lot of materials for your questions in the process of writing the paper, and also providing enthusiastic help in the process of writing and typesetting the paper. 4 CONCLUSION 8. Yin Chengguo: Problems and countermeasures in the development of small and medium-sized enterprises in the Pearl River Delta [J]. Journal of Wuyi University (Social Science Edition), 2002 (03): 1-4+29. According to the survey statistics and related data of the Guangdong Provincial Government in 2017, the article analyzes the financing difficulties and brain drain of small and medium-sized enterprises in the development process of the Pearl River Delta region by reading relevant materials and literature, and gives corresponding countermeasures. After the implementation of strategies, the corresponding problems can be solved to promote the economic development in China. 5. Wu Dequn. The transformation and upgrading of SMEs is imminent [N]. Shenzhen Special Zone News, 2012-08-24 (A06). REFERENCES 1. Li Yifu. Research on the influence of Guangdong SME clusters on economic development [J]. Times Economics and Trade, 2015(17): 104-106. 2. Yuan Youjun. The current situation and development thinking of small and medium enterprises in Guangdong [J]. Import and export manager, 2014(S1): 47-48. 2. Yuan Youjun. The current situation and development thinking of small and medium enterprises in Guangdong [J]. Import and export manager, 2014(S1): 47-48. 2. Yuan Youjun. The current situation and development thinking of small and medium enterprises in Guangdong [J]. Import and export manager, 2014(S1): 47-48. 3. Liu Ji. Research on the Development of Small and Medium-sized Enterprises in Guangdong and Government Behavior [D]. Shaanxi Normal University, 2013. 4. Feng Dechong. The dilemma and outlets of Shenzhen's small and medium-sized enterprises[J]. Special Zone Practice and Theory, 2013(04): 54-56+60. 5. Wu Dequn. The transformation and upgrading of SMEs is imminent [N]. Shenzhen Special Zone News, 2012-08-24 (A06). 5. Wu Dequn. The transformation and upgrading of SMEs is imminent [N]. Shenzhen Special Zone News, 2012-08-24 (A06). 4
https://openalex.org/W2892129123	https://europepmc.org/articles/pmc6151259?pdf=render	English	null	Effect of floods on the δ<sup>13</sup>C values in plant leaves: a study of willows in Northeastern Siberia	PeerJ	2,018	cc-by	10,578	ABSTRACT Although stable carbon isotopic composition (d13C) of plants has been widely used to indicate different water regimes in terrestrial ecosystems over the past four decades, the changes in the plant d13C value under waterlogging have not been sufﬁciently clariﬁed. With the enhanced global warming in recent years, the increasing frequency and severity of river ﬂoods in Arctic regions lead to more waterlogging on willows that are widely distributed in river lowland. To investigate the d13C changes in plants under different water conditions (including waterlogging), we measured the d13C values in the leaves of willows with three species, Salix boganidensis, S. glauca, and S. pulchra, and also monitored changes in plant physiology, under several major ﬂooding conditions in Northeastern Siberia. The foliar d13C values of willows varied, ranging from -31.6 to -25.7‰ under the different hydrological status, which can be explained by: (i) under normal conditions, the foliar d13C values decrease from dry (far from a river) to wet (along a river bank) areas; (ii) the d13C values increase in frequently waterlogged areas owing to stomatal closure; and (iii) after prolonged ﬂooding periods, the d13C values again decrease, probably owing to the effects of not only the closure of stomata but also the reduction of foliar photosynthetic ability under long period of waterlogging. Based on these results, we predict that plant d13C values are strongly inﬂuenced by plant physiological responses to diverse hydrological conditions, particularly the long periods of ﬂooding, as occurs in Arctic regions. Submitted 3 April 2018 Accepted 16 July 2018 Published 20 September 2018 Corresponding authors Rong Fan, fanrong@ees.hokudai.ac.jp Atsuko Sugimoto, atsukos@ees.hokudai.ac.jp Academic editor Miquel Gonzalez-Meler Additional Information and Declarations can be found on page 17 DOI 10.7717/peerj.5374 Copyright 2018 Fan et al. Distributed under Creative Commons CC-BY 4.0 Submitted 3 April 2018 Accepted 16 July 2018 Published 20 September 2018 Corresponding authors Rong Fan, fanrong@ees.hokudai.ac.jp Atsuko Sugimoto, atsukos@ees.hokudai.ac.jp Academic editor Miquel Gonzalez-Meler Additional Information and Declarations can be found on page 17 DOI 10.7717/peerj.5374 Copyright 2018 Fan et al. Distributed under Creative Commons CC-BY 4.0 Subjects Climate Change Biology, Biogeochemistry, Ecohydrology Keywords Siberia, Stable carbon isotope, Willows, River lowland, Photosynthesis, Waterlogging, Flooding, Stomatal regulation How to cite this article Fan et al. (2018), Effect of ﬂoods on the d13C values in plant leaves: a study of willows in Northeastern Siberia. PeerJ 6:e5374; DOI 10.7717/peerj.5374 Effect of ﬂoods on the δ13C values in plant leaves: a study of willows in Northeastern Siberia Rong Fan1, Tomoki Morozumi1, Troﬁm C. Maximov2,3 and Rong Fan1, Tomoki Morozumi1, Troﬁm C. Maximov2,3 and Atsuko Sugimoto3,4,5,6 1 Graduate School of Environmental Science, Hokkaido University, Sapporo, Hokkaido, Jap 1 Graduate School of Environmental Science, Hokkaido University, Sapporo, Hokkaido, Japan 2 Institute for Biological Problems of Cryolithozone, Siberian Blanch of Russian Academy of Sciences, Yakutsk, Sakha, Russia 3 North Eastern Federal University, Yakutsk, Sakha, Russia 4 y 4 Arctic Research Center, Hokkaido University, Sapporo, Hokkaido, Japan 5 5 Global Station for Arctic Research, Global Institution for Collaborative Research and Education, Hokkaido University, Sapporo, Hokkaido, Japan 6 6 Faculty of Environmental Earth Science, Hokkaido University, Sapporo, Hokkaido, Japan INTRODUCTION Over the past four decades, stable carbon isotopic composition (d13C, ‰ relative to Vienna Pee Dee Belemnite, VPDB) of plants has been widely employed as a conventional tool to estimate changes in carbon ﬂux as plant physiology responds to environmental Distributed under Creative Commons CC-BY 4.0 changes. The magnitude of isotopic fractionation is indeed highly dependent on physiological conditions (Farquhar, Ehleringer & Hubick, 1989; Robinson, 2001). For instance, it is well known that carbon isotopic fractionation (D13C) in plants is a function of the ratio of leaf intercellular-(ci) to atmospheric (ca) CO2 concentrations (ci/ca) (Farquhar & Sharkey, 1982; Farquhar, Ehleringer & Hubick, 1989), as given in Eq. (1): 13C ¼ d13Ca d13Cp ¼ a þ b a ð Þ Ci Ca ; (1) D13C ¼ d13Ca d13Cp ¼ a þ b a ð Þ Ci Ca ; (1) where, d13Ca and d13Cp are the d13C values of atmospheric CO2 and photosynthate, respectively; while a and b are the carbon isotopic fractionations associated with CO2 diffusion and enzymatic carboxylation (carbon ﬁxation) in plant leaves, respectively. The ci/ca ratio is usually determined from the balance between the CO2 supply controlled by stomatal conductance and CO2 consumption via the carboxylation related to photosynthetic activity. When the stomata closes (e.g., in response to a large water deﬁcit and high evaporation rates due to high ambient temperature, (Meidner & Mansﬁeld, 1968; Willmer & Fricker, 1996)), low CO2 supply reduces ci, leading to a decrease in the D13C values and, ultimately, an increase in the d13Cp values. On the other hand, when CO2 consumption decreases as a consequence of reducing photosynthetic activity (e.g., due to the limitations of light and nutrients (Hall & Krishna, 1999)), a large ci increases the D13C values and, ultimately, decreases the d13Cp values. These effects are expressed by Eq. (2), A gc ¼ Ca Ci ¼ Ca 1 Ci Ca ; (2) (2) where A is the photosynthetic rate, gc is the stomatal conductance of CO2, and gs is stomatal conductance which equals 1.6 times gc. where A is the photosynthetic rate, gc is the stomatal conductance of CO2, and gs is stomatal conductance which equals 1.6 times gc. Combining Eqs. (1) and (2), the D13C and d13Cp values are given by the standard Eq. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 INTRODUCTION (3): D13C ¼ d13Ca d13Cp ¼ b b a Ca 1:6A gs ; (3 (3) Thus, the plant d13C values are primarily controlled by both stomatal conductance (gs) for CO2 and photosynthetic activity (A) (Farquhar & Richards, 1984). For example, under constant A, the d13Cp values are controlled mainly by the gs. Drought-induced low gs decreases the D13C values and increases the d13Cp values. In contrast, moisture-induced high gs increases the D13C values and decreases the d13Cp values (Farquhar & Richards, 1984; Knight, Livingston & Van Kessel, 1994; Korol et al., 1999; Barbour & Farquhar, 2000; Warren, McGrath & Adams, 2001; Huang et al., 2008; Peri et al., 2012). Under constant gs, however, the d13Cp values are primarily controlled by A, which is strongly correlated with light intensity (Yakir & Israeli, 1995) and nutrient availability (Ripullone et al., 2004; Duursma & Marshall, 2006; Kranabetter et al., 2010). Enhanced A decreases the D13C values and, ultimately, increases the d13Cp values (O’Leary, 1988; Farquhar, Ehleringer & Hubick, 1989). Thus, the plant d13C values are primarily controlled by both stomatal conductance (gs) for CO2 and photosynthetic activity (A) (Farquhar & Richards, 1984). For example, Thus, the plant d13C values are primarily controlled by both stomatal conductance (gs) for CO2 and photosynthetic activity (A) (Farquhar & Richards, 1984). For example, Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Figure 1 Schematic view of the possible foliar d13C values under various hydrological conditions. gs and A are stomatal conductance and photosynthetic activity, respectively. Dry and wet are without waterlogging, and waterlogging and long period waterlogging represent continual and continuous waterlogging, respectively. Possible changes in the foliar d13C value are shown for assumed scenarios (S1, S2, and S3). Full-size  DOI: 10.7717/peerj.5374/ﬁg-1 Figure 1 Schematic view of the possible foliar d13C values under various hydrological conditions. gs and A are stomatal conductance and photosynthetic activity, respectively. Dry and wet are without waterlogging, and waterlogging and long period waterlogging represent continual and continuous waterlogging, respectively. Possible changes in the foliar d13C value are shown for assumed scenarios (S1, S2, and S3). Full-size  DOI: 10.7717/peerj.5374/ﬁg-1 Figure 1 Schematic view of the possible foliar d13C values under various hydrological conditions. gs and A are stomatal conductance and photosynthetic activity, respectively. Dry and wet are without waterlogging, and waterlogging and long period waterlogging represent continual and continuous waterlogging, respectively. INTRODUCTION Possible changes in the foliar d13C value are shown for assumed scenarios (S1, S2, and S3). Full-size  DOI: 10.7717/peerj.5374/ﬁg-1 With respect to the river ﬂooding, there is physiological evidence that stomata can also be closed in response to waterlogging conditions (Gomes & Kozlowski, 1980; Olivella et al., 2000; Copolovici & Niinemets, 2010); though, such evidence does not include isotope data, such as the D13C or d13Cp values. If the CO2 gs term in Eq. (3) decreases due to the low stomatal conductance during waterlogging, low foliar D13C (high foliar d13Cp) values will appear very similar to the values observed under drought conditions. Indeed, previous studies have reported changes in the d13Cp value under both, natural and simulated waterlogging conditions. Anderson et al. (2005) found that tree-ring d13C values for the pond cypress Taxodium ascendens in their natural environments are positively correlated with the total annual precipitation; similarly, Li & Sugimoto (2017) reported an increase in needle d13Cp values for the larch Larix gmelinii in waterlogging pot experiments. The latter study attributed the increase in larch needle d13Cp values to low gs caused by waterlogging. Although Anderson et al. (2005) reported a decreased gs with an increased A under very wet conditions, most physiological experiments have demonstrated that not only gs, but also A is apparently reduced under waterlogging (Gomes & Kozlowski, 1980; Copolovici & Niinemets, 2010; Li & Sugimoto, 2017). Based on these ﬁndings, we hypothesize that the D13C (and d13Cp) values in plant leaves are not exclusively under the controlling of stomata (gs), because the photosynthetic rate (A) is also not constant in waterlogging. Moreover, net A and chlorophyll contents were observed decreasing without any change in either gs or ci/ca, in a continuous waterlogging experiment with okra Abelmoschus esculentus (Ashraf & Arfan, 2005), a waterlogging-tolerant plant. Thus, the possible changes in the foliar d13C value under long period (continuous) waterlogging, which are assumed more dependent on changes of photosynthetic rate, are shown in Fig. 1 under predicted scenarios 1, 2, and 3 (S1, S2, and S3). Moreover, it is possible, as S3, that under low gs, a reduction of A can lead to lower d13Cp values. Thus, D13C (and d13Cp) Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 3/20 values will be potentially changed in terms of frequency, magnitude, and duration of waterlogging. values will be potentially changed in terms of frequency, magnitude, and duration of waterlogging. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 INTRODUCTION The Arctic region is highly sensitive and responsive to climatic changes (Giorgi, 2006). Thus, increases in atmospheric temperature signiﬁcantly affect the hydrology in this region, including prevailing spring ﬂoods (Shahgedanova, 2002; Shiklomanov et al., 2007; Tan, Adam & Lettenmaier, 2011). For example, with rising temperature, the annual average discharge rate from the 19 largest rivers in the Arctic increased by approximately 10% from 1977 to 2007 (Overeem & Syvitski, 2010). Since the topography of the Arctic river lowlands is relatively ﬂat, spring ﬂooding strongly inﬂuences riparian plant communities. Shrubs which can stand high moisture levels, predominate over low moisture-preferring trees like larch and pine in areas along rivers under recurrent spring ﬂoods (Troeva et al., 2010). For instance, in the wide Indigirka River lowland near Chokurdakh village Russia in Northeastern Siberia, one sixth of a 10 10 km2 area is covered by dwarf shrub willow (Salix) (T. Morozumi, 2015, personal communication) and particularly being abundant on river banks. Thus, because willows in this area are exposed to an increase frequency of river ﬂoods and have high chances to be submerged, they are a good candidate species to study the effects of ﬂooding on the d13Cp values of leaves in relation to A and gs. The objective of this study was to determine the effects of ﬂooding on the d13C values in willow leaves under four major hydrological conditions: dry, wet, and short and long period waterlogging (Fig. 1). We measured the d13C values of bulk leaves from willows growing under these ﬂooding regimes in the Indigirka River lowland of Northeastern Siberia. MATERIALS AND METHODS Study area The study site is located in the Indigirka River lowland near Chokurdakh (7038′N, 14753′E), Sakha Republic (Yakutia), Russian Federation (Fig. 2). Mean annual air temperature in the region between 1950 and 2016 was -13.7 C, ranging from -33.9 C in January (the coldest month) to 10.1 C in July (the warmest month). Mean annual precipitation between 1950 and 2008 was 209 mm year-1 (Yabuki et al., 2011). p p y ( , ) The Indigirka River lowland, including rivers, lakes, wetlands, hills, and ﬂoodplains, is frequently ﬂooded during spring and summer. Soils in the region are loamy or silty-loamy alluvial soils with black- to grayish-olive color along the riverbanks (Troeva et al., 2010). The average depth of the active layer in soils is approximately 30 cm on land and one m near the river in the summer. The local vegetation consists of aquatics, sphagnums mosses, graminoids, shrubs (mainly the willow Salix sp. and the dwarf birch Betula nana), alders, larches, and pines. Between 1970 and 2016, the average intra-annual water level cycle of the Indigirka River was 70 ± 83 mm for April and May (late winter, pre-ﬂooding), increasing to 600 ± 93 mm for June–August (spring and summer, ﬂooding season); then, gradually receding to 343 ± 146 mm for September and October (autumn and early winter, post-ﬂooding), and declining further to 56 ± 26 mm in winter (after October). Field experiments were approved by Hokkaido University, and Institute for Biological Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Figure 2 Sampling sites and schematic illustration of a transect. (A) Sampling sites near Chokurdakh village in the study region, northeastern Siberia. Thick and thin blue lines represent the Indigirka River and its tributaries, respectively. Areas ﬁlled with light blue represent lakes. Triangles (18), stars (3), ﬁlled black circles (3) and empty circle (1) indicate the sampling sites, three transects (SKA, SKB, and SBoydom), three sites for production measurement (LAI1∼3) and one site for photosynthesis monitoring (SPh). More sampling sites see Table A2. (B) A schematic illustration of a transect. Full-size  DOI: 10.7717/peerj.5374/ﬁg-2 Figure 2 Sampling sites and schematic illustration of a transect. (A) Sampling sites near Chokurdakh village in the study region, northeastern Siberia. Thick and thin blue lines represent the Indigirka River and its tributaries, respectively. Areas ﬁlled with light blue represent lakes. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Study area Triangles (18), stars (3), ﬁlled black circles (3) and empty circle (1) indicate the sampling sites, three transects (SKA, SKB, and SBoydom), three sites for production measurement (LAI1∼3) and one site for photosynthesis monitoring (SPh). More sampling sites see Table A2. (B) A schematic illustration of a transect. Full-size  DOI: 10.7717/peerj.5374/ﬁg-2 Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Problems of Cryolithozone, Siberian Branch of Russian Academy of Science, and North-Eastern Federal University. Willows in the Indigirka River lowland The common willow species observed in 2015–2017 were Salix boganidensis, S. pulchra, S. glauca, S. richardsonii, S. viminalis, S. alaxensis, S. fuscescens, and S. hastata. Most species were 1 m tall, except for a few species such as S. boganidensis, S. alaxensis, and S. fuscescens, which were two to three m in height. Diameter at breast height generally ranged between one and six cm. Maximum root depth was approximately one m at the riverbank, but was highly variable and depended on various factors such as the thickness of the active soil layers and moisture levels where the willows grew. Willows were distributed more densely along the riverbanks than on dry lands. Observations conducted with a GardenWatchCam time-lapse camera (Brinno, Inc., Taipei City, Taiwan) showed that the buds of willow leaves opened around the ﬁrst few weeks of June, when the snow had melted and the daily average air temperature had increased to >0 C. The leaves and stems grew rapidly, within 10 days after bud opening, and were fully developed by mid-July. Willow leaf biomass peaked by the end of July, and this observation was consistent with a normalized difference vegetation index (NDVI) study in Alaska (Boelman et al., 2011). Aboveground net primary production (ANPP, newly formed stems and leaves in each year) and the leaf area index (LAI) of the willows in 2016 were measured using the direct harvesting method (Jonckheere et al., 2004) in three blocks which were predominated by willows. ANPP was 63, 119, and 117 g m-2·a in each of the three blocks, and the LAI was 0.59, 0.71, and 1.59 in each of the three blocks (Table A1). Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Samples In the summer of 2015 and 2016, we collected leaves from the locally dominant willows Salix boganidensis, S. glauca, and S. richardsonii on three sets of 20 m transects (SBoydom, SKA, and SKB) from the river. SBoydom is located between the mainstream Indigirka and the wetland; while, SKA and SKB are situated next to a secondary tributary, Kryvaya (Fig. 2; Table A2). Three points, named PA, PB, and PC, were marked on each transect based on their distance to the river. The maximum thaw depth was always found at PA. This layout was designed based on the differences in intra- and inter-annual ﬂooding conditions (Figs. 2 and 3). PAs at SKB and SBoydom were continually waterlogged throughout the growing season in 2015 and continuously waterlogged until July in 2016 (Fig. 3). PB at SKB and PA at SKA were ﬂooded only in 2016 (Fig. 3). Four current-year top shoots were collected at each point at the end of the growing season (the end of July) in both 2015 and 2016. Current-year shoots were also randomly sampled from willows in local scale on the Indigirka River lowland during the same period. A total of 31 sites with different locations were used in 2015 and 2016 (Fig. 2; Table A2). At least four current-year shoots were collected at each location to obtain representative data for each site. The details of sampling sites, locations, species, and sampling numbers are shown in Fig. 2 and Table A2. All samples were immediately dried at 60 C for 48 h after collecting. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 6/20 Figure 3 Schematic view of each transect, possible changes in the hydrological conditions, and foliar d13C values (‰). (A) Schematic view of each transect with the highest water levels observed in each case in 2015 (blue) and 2016 (light blue) and the height of PB and PC, compared to PA (black line). (B) Possible changes (black arrows) in the hydrological conditions from 2015 (ﬁlled circles) to 2016 (open triangles) in each point on transects. Dry and wet are without waterlogging, and WL and LWL represent waterlogging (continual) and long period waterlogging (continuous), respectively. (C) The foliar d13C values (‰) found in willows were reported as mean ± SD. Full-size  DOI: 10.7717/peerj.5374/ﬁg-3 Figure 3 Schematic view of each transect, possible changes in the hydrological conditions, and foliar d13C values (‰). Samples (A) Schematic view of each transect with the highest water levels observed in each case in 2015 (blue) and 2016 (light blue) and the height of PB and PC, compared to PA (black line). (B) Possible changes (black arrows) in the hydrological conditions from 2015 (ﬁlled circles) to 2016 (open triangles) in each point on transects. Dry and wet are without waterlogging, and WL and LWL represent waterlogging (continual) and long period waterlogging (continuous), respectively. (C) The foliar d13C values (‰) found in willows were reported as mean ± SD. Full-size  DOI: 10.7717/peerj.5374/ﬁg-3 Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Photosynthetic rate and stomatal conductance analyses Supporting data on the foliar d13C values, the photosynthetic rate and stomatal conductance of willow leaves were monitored in the ﬁeld in 2017 using a portable porometer (LCpro+; ADC BioScientiﬁc Ltd, Hoddesdon, Herts, UK) equipped with a conifer chamber and a lighting system. The photosynthetic rate (A) of S. boganidensis, S. richardsonii, and S. glauca under different light levels (10–955 mmol m-2 s-1) was measured to obtain light response curves and thus, to identify the saturation light intensity. Supporting data on the foliar d13C values, the photosynthetic rate and stomatal conductance of willow leaves were monitored in the ﬁeld in 2017 using a portable porometer (LCpro+; ADC BioScientiﬁc Ltd, Hoddesdon, Herts, UK) equipped with a conifer chamber and a lighting system. The photosynthetic rate (A) of S. boganidensis, S. richardsonii, and S. glauca under different light levels (10–955 mmol m-2 s-1) was measured to obtain light response curves and thus, to identify the saturation light intensity. Site SPh near Chokurdakh village, was set up in the summer of 2017 to monitor the conditions in former transects SKA, SKB, and SBoydom, since the extremely high ﬂooding caused all these three sites totally submerged for the entire summer of 2017. Under gradient ﬂooding conditions on site SPh (-PA: submerged till July 20; -PB: submerged till July 15; -PC: without submergence during the observation period), temporary changes were measured in the photosynthetic rate (A) and stomatal conductance (gs) of S. richardsonii, S. glauca, and S. boganidensis in response to a single saturated light exposure at 600 mmol m-2 s-1 around noon, the rest of chamber conditions were set to match ambient conditions. For each measurement at the points (PA, PB, or PC), a total of 12 leaves from four trees were marked for leaf ADC data recording for more than six times on any leaf of them. Average of all records was calculated for each measurement. Measurements were taken ﬁve times every 2–3 days between July 13, 2017 and July 27, 2017. The leaves were also collected after whole monitoring period to check the foliar d13C values. Site SPh near Chokurdakh village, was set up in the summer of 2017 to monitor the conditions in former transects SKA, SKB, and SBoydom, since the extremely high ﬂooding caused all these three sites totally submerged for the entire summer of 2017. Stable carbon isotope analysis Dried leaves were milled into ﬁne powder with liquid N2 and dried again at 60 C for 48 h; each sample was then wrapped in a tin capsule and injected into an elemental analyzer (Flash EA 1112; Thermo Fisher Scientiﬁc, Bremen, Germany), connected to an isotope ratio mass spectrometry (IRMS, Delta V; Thermo Fisher Scientiﬁc, Bremen, Germany) through a continuous-ﬂow carrier-gas system (Conﬂo III; Thermo Fisher Scientiﬁc, Bremen, Germany). The stable carbon isotopic composition was reported in the standard d notation relative to VPDB. A laboratory standard was injected after every ten samples Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 7/20 to verify that the analytical accuracy was better than 0.1‰. To reduce the effect of sampling heterogeneity in d13C within a single site, four samples were measured and the average isotopic composition was reported for each site, with the standard deviation ranging from 0.0 to 1.4‰ (average, 0.7‰). Photosynthetic rate and stomatal conductance analyses Under gradient ﬂooding conditions on site SPh (-PA: submerged till July 20; -PB: submerged till July 15; -PC: without submergence during the observation period), temporary changes were measured in the photosynthetic rate (A) and stomatal conductance (gs) of S. richardsonii, S. glauca, and S. boganidensis in response to a single saturated light exposure at 600 mmol m-2 s-1 around noon, the rest of chamber conditions were set to match ambient conditions. For each measurement at the points (PA, PB, or PC), a total of 12 leaves from four trees were marked for leaf ADC data recording for more than six times on any leaf of them. Average of all records was calculated for each measurement. Measurements were taken ﬁve times every 2–3 days between July 13, 2017 and July 27, 2017. The leaves were also collected after whole monitoring period to check the foliar d13C values. Statistical analysis Linear Mixed Models (LMMs) were used to clarify differences in the foliar d13C value among willows growing in three transects in 2015 and 2016. Foliar d13C value was set as the response variable, with ﬂooding condition was set as the ﬁxed effect, and species (i.e., S. boganidensis, S. richardsonii, and S. glauca) was set as a random effect. Similar analyses by LMMs were also used to ﬁgure out any differences in the foliar d13C value among the willows randomly collected on the Indigirka River lowland in 2015 and 2016. Foliar d13C value was set as the response variable, the ﬂooding condition was assigned as the ﬁxed effect, and the location (along the mainstream or the tributary), and species (Table A2), were set as random effects. Tukey’s test was used as a post hoc analysis for multiple comparisons. The lme4 package (Bates, Maechler & Walker, 2015) of R (R Core Team, 2015) was used to build the LMMs. RESULTS Foliar d13C in the transects Foliar d13C values differed among SKA, SKB, and SBoydom, and between years (Fig. 3). Along transect SKA in 2015, the mean foliar d13C values were -30.3 ± 0.8‰ for PA Foliar d13C values differed among SKA, SKB, and SBoydom, and between years (Fig. 3). Along transect SKA in 2015, the mean foliar d13C values were -30.3 ± 0.8‰ for PA Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Figure 4 Statistical analysis for the foliar d13C values (‰) under four different hydrological conditions in transects in 2015 and 2016. Box-and-whisker plot of the statistical analysis for the foliar d13C values (‰) under four different hydrological conditions in sampling transects established in year 2015 and 2016. Different letters over the numbers indicate statistically signiﬁcant differences according to Turkey’s post hoc test and Linear Mixed Model. Dry and wet are without waterlogging, and WL and LWL represent waterlogging (continual) and long period waterlogging (continuous), respectively. Full-size  DOI: 10.7717/peerj.5374/ﬁg-4 Figure 4 Statistical analysis for the foliar d13C values (‰) under four different hydrological conditions in transects in 2015 and 2016. Box-and-whisker plot of the statistical analysis for the foliar d13C values (‰) under four different hydrological conditions in sampling transects established in year 2015 and 2016. Different letters over the numbers indicate statistically signiﬁcant differences according to Turkey’s post hoc test and Linear Mixed Model. Dry and wet are without waterlogging, and WL and LWL represent waterlogging (continual) and long period waterlogging (continuous), respectively. Full-size  DOI: 10.7717/peerj.5374/ﬁg-4 Figure 4 Statistical analysis for the foliar d13C values (‰) under four different hydrological conditions in transects in 2015 and 2016. Box-and-whisker plot of the statistical analysis for the foliar d13C values (‰) under four different hydrological conditions in sampling transects established in year 2015 and 2016. Different letters over the numbers indicate statistically signiﬁcant differences according to Turkey’s post hoc test and Linear Mixed Model. Dry and wet are without waterlogging, and WL and LWL represent waterlogging (continual) and long period waterlogging (continuous), respectively. Full-size  DOI: 10.7717/peerj.5374/ﬁg-4 (close to the river but without submergence), which was much lower than those for PB (-28.0 ± 0.6‰) and PC (-27.5 ± 0.7‰). Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 RESULTS Box-and-whisker plot of the statistical analysis for the foliar d13C values (‰) under different environments with distinct hydrological conditions at local scale in year 2015 (A) and 2016 (B), different letters over the numbers indicate statistically signiﬁcant differences according to Tukey’s post hoc test and the Linear Mixed Model. Full-size  DOI: 10.7717/peerj.5374/ﬁg-5 hydrological conditions (i.e., “Dry,” “Wet,” “WL,” and “LWL,” in Figs. 3 and 4). The foliar d13C values were high in dry and waterlogged continually conditions. Conversely, values under wet and continuous long period waterlogging were consistently low. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 RESULTS (2018), PeerJ, DOI 10.7717/peerj.5374 Figure 5 Statistical analysis for the foliar d13C values (‰) under different hydrological conditions at local scale in 2015 and 2016. Box-and-whisker plot of the statistical analysis for the foliar d13C values (‰) under different environments with distinct hydrological conditions at local scale in year 2015 (A) and 2016 (B), different letters over the numbers indicate statistically signiﬁcant differences according to Tukey’s post hoc test and the Linear Mixed Model. Full-size  DOI: 10.7717/peerj.5374/ﬁg-5 hydrological conditions (i.e., “Dry,” “Wet,” “WL,” and “LWL,” in Figs. 3 and 4). The foliar d13C values were high in dry and waterlogged continually conditions. Conversely, values under wet and continuous long period waterlogging were consistently low. Figure 5 Statistical analysis for the foliar d13C values (‰) under different hydrological conditions at local scale in 2015 and 2016. Box-and-whisker plot of the statistical analysis for the foliar d13C values (‰) under different environments with distinct hydrological conditions at local scale in year 2015 (A) and 2016 (B), different letters over the numbers indicate statistically signiﬁcant differences according to Tukey’s post hoc test and the Linear Mixed Model. Full-size  DOI: 10.7717/peerj.5374/ﬁg-5 Figure 5 Statistical analysis for the foliar d13C values (‰) under different hydrological conditions at local scale in 2015 and 2016. Box-and-whisker plot of the statistical analysis for the foliar d13C values (‰) under different environments with distinct hydrological conditions at local scale in year 2015 (A) and 2016 (B), different letters over the numbers indicate statistically signiﬁcant differences according to Tukey’s post hoc test and the Linear Mixed Model. Full-size  DOI: 10.7717/peerj.5374/ﬁg-5 Figure 5 Statistical analysis for the foliar d13C values (‰) under different hydrological conditions at local scale in 2015 and 2016. Box-and-whisker plot of the statistical analysis for the foliar d13C values (‰) under different environments with distinct hydrological conditions at local scale in year 2015 (A) and 2016 (B), different letters over the numbers indicate statistically signiﬁcant differences according to Tukey’s post hoc test and the Linear Mixed Model. Full-size  DOI: 10.7717/peerj.5374/ﬁg-5 hydrological conditions (i.e., “Dry,” “Wet,” “WL,” and “LWL,” in Figs. 3 and 4). The foliar d13C values were high in dry and waterlogged continually conditions. Conversely, values under wet and continuous long period waterlogging were consistently low. Figure 5 Statistical analysis for the foliar d13C values (‰) under different hydrological conditions at local scale in 2015 and 2016. RESULTS A similar trend was also observed in 2016, when the water level was high; in this case, the foliar d13C values for PA (-29.4 ± 0.4‰) was lower than those for PB (-29.2 ± 0.3‰) and PC (-28.6 ± 0.9‰); although, the difference was small. For the inter-annual changes from 2015 to 2016 on transect SKA, an increase in foliar d13C value was observed for PA (+0.8 ± 0.6‰); whereas, a decrease was recorded for both, PB (-1.2 ± 0.5‰) and PC (-1.0 ± 0.8‰). In 2015, the sampling point at PAs along both, the SKB and SBoydom transects, were sometimes waterlogged (“WL”; Fig. 3); whereas, all points PBs and PCs were not. A similar trend for the foliar d13C values was found in both, SKB and SBoydom transects, as the foliar d13C values for PAs (-27.4 ± 1.1‰ and -27.3 ± 0.7‰, respectively) were higher than those for PBs (-28.5 ± 0.6‰ and -27.4 ± 1.0‰, respectively), and PCs (-28.1 ± 0.9‰ and -28.3 ± 0.7‰, respectively) (Fig. 3). In 2016, although PAs were also but always waterlogged (“LWL”; Fig. 3), PBs and PCs were not, trends in the foliar d13C value were apparently different between SKB and SBoydom. In SKB, the foliar d13C values for PA (-29.0 ± 0.6‰) were slightly higher than those for PB (-29.2 ± 0.5‰) and PC (-29.7 ± 0.9‰); whereas, in SBoydom, values for PA (-29.5 ± 0.7‰) were slightly lower than those for PB (-28.3 ± 0.5‰) and PC (-28.0 ± 0.8‰) (Fig. 3). For the inter-annual changes in SKB and SBoydom, decreases in the foliar d13C value were observed at all points in all transects, except for PC in SBoydom. The differences in the foliar d13C value between the PB and PC within each transect ranging from -0.5 to +0.9‰; however, those for the PA signiﬁcantly deviated (-2.5 to +0.9‰) from the mean value of the PB and PC. Overall, statistical analysis of the foliar d13C values from transects (in Fig. 3) showed a signiﬁcant difference (F3,42 = 42.276, P < 0.01, Fig. 4) among the four major Overall, statistical analysis of the foliar d13C values from transects (in Fig. 3) showed a signiﬁcant difference (F3,42 = 42.276, P < 0.01, Fig. 4) among the four major Fan et al. DISCUSSION River water level and leaf formation Photosynthetic rate and stomatal conductance Photosynthetic rate (A) gradually increased asymptotically and reached to about six mmol m-2 s-1 for S. richardsonii, about six to eight mmol m-2 s-1 for S. glauca, and about 8–12 mmol m-2 s-1 for S. boganidensis, under 400–600 mmol m-2 s-1 (irradiation scanning covered the range from 10 to 955 mmol m-2 s-1) (Fig. A1). Therefore, the saturating light intensity for willow leaves in the Indigirka River lowland was found in the range from 400 to 600 mmol m-2 s-1. Maximum photosynthetic rate A recorded in leaves of S. boganidensis was the highest among all three species (Fig. A1). In the summer of 2017, willows at the SPh-PA were continuously submerged until July 20; willows at the SPh-PB had just come out of the water when monitoring began on July 15; while, willows at the SPh-PC were not submerged during the monitoring period (Fig. 6A), although all three points at transect SPh were within 20 m from the river. The largest decrease (-1.6 ± 1.4 mmol m-2 s-1) in A at SPh-PA during the monitoring period, was registered on July 21 when the waterlogging just ﬁnished; whereas, after ﬂooding A was observed to follow a slow recovery over the last few days (Fig. 6B). A similar decrease-increase trend in A was also detected in the willow leaves at SPh-PB, and also, in this case, the lowest A was recorded soon after waterlogging ﬁnished on July 18 (-0.7 ± 2.6 mmol m-2 s-1 lower than ﬁrst measurement) (Fig. 6B). However, at SPh-PC, A continuously increased, compared to the initial measurement. These values corresponded with the waterlogging gradients in SPh, that A was reduced under waterlogging and could recover if waterlogging was over. On the other hand, among points, compared to SPh-PC, the lowest gs values were found at SPh-PA; while, intermediate values were recorded in SPh-PB (Fig. 6C). Thus, gs, apparently correlated to the degree of waterlogging among SPh-PA, -PB, and -PC. Spatial distribution in the foliar δ13C of willows Neither signiﬁcant nor large differences were detected in the foliar d13C value in any of the willows growing in the 31 randomly selected sampling sites in local scale (with different locations) in Indigirka River lowland during the same sampling periods at the end of the growing seasons of 2015 and 2016 (Fig. 5). The willow foliar d13C values ranged from -31.1 to -25.3‰ in 2015, and from -31.6 to -25.7‰ in 2016. Statistical analysis shows that there were no signiﬁcant differences between the four environmental conditions (ﬂooding, ﬂooded, on land, and wetland) in 2015 or 2016. Moreover, the d13C values measured for willow leaves collected in a larch forest in 2016 (-27.6 ± 1.2‰) were signiﬁcantly higher than those sampled anywhere else in the same year (F4 168 = 2.58, P = 0.039). Neither signiﬁcant nor large differences were detected in the foliar d13C value in any of the willows growing in the 31 randomly selected sampling sites in local scale (with different locations) in Indigirka River lowland during the same sampling periods at the end of the growing seasons of 2015 and 2016 (Fig. 5). The willow foliar d13C values ranged from -31.1 to -25.3‰ in 2015, and from -31.6 to -25.7‰ in 2016. Statistical analysis shows that there were no signiﬁcant differences between the four environmental conditions (ﬂooding, ﬂooded, on land, and wetland) in 2015 or 2016. Moreover, the d13C values measured for willow leaves collected in a larch forest in 2016 (-27.6 ± 1.2‰) were signiﬁcantly higher than those sampled anywhere else in the same year (F4,168 = 2.58, P = 0.039). Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 River water level and leaf formation As mentioned before, the willow leaves began opening after the ﬁrst week in June and ﬁnished growing by the end of July. This suggests that the foliar d13C values of willow leaves recorded hydrological conditions experienced from early mid-June to the date of collection, which is a longer period than that of the in situ observation and monitoring period for hydrological conditions. However, it is known that the river water level is gradually decreased but by within approximately one m during the term of leaf growing (Fig. A2). Moreover, only small differences were found in the foliar d13C values between top and bottom of a single current-year shoot, approximately 0.5 ± 0.1‰ (Fig. A3), which may have experienced leaf formation over different periods. Thus, in present ﬁeld observation study, the hydrological conditions observed during July are assumed to be almost the same or very similar to those for the early part of the growing season. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Foliar δ13C values under different hydrological conditions Foliar δ13C values in normal dry-wet conditions In the normal dry-wet SKA transect, in the absence of waterlogging during 2015, S. boganidensis grew at PA with more available water than those at PB or PC. The willow Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Figure 6 Hydrological conditions in SPh, and results of physiology monitoring. (A) Hydro conditions in SPh-PA (circles), -PB (triangles), and -PC (squares), with the period of submergence in blue shaded bars and relative foliar d13C values (‰), and (B) photosynthetic rate (A, mmol m-2s- (C) stomatal conductance (gs, mol m-2s-1) with ﬁxed radiation (600 mmol m-2s-1), during July 13th Mean ± SD. Full-size  DOI: 10.7717/peerj.537 Figure 6 Hydrological conditions in SPh, and results of physiology monitoring. (A) Hydrological conditions in SPh-PA (circles), -PB (triangles), and -PC (squares), with the period of submergence shown in blue shaded bars and relative foliar d13C values (‰), and (B) photosynthetic rate (A, mmol m-2s-1) and (C) stomatal conductance (gs, mol m-2s-1) with ﬁxed radiation (600 mmol m-2s-1), during July 13th–27th. Mean ± SD. Full-size  DOI: 10.7717/peerj.5374/ﬁg-6 12/20 Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 leaves at PA were largely depleted in 13C, with the difference of d13C value by about 2‰, relative to those at PB or PC (Fig. 3). These results are consistent with the well-known fact that, under dry conditions, low gs results in high foliar d13C values; whereas, under wet conditions, high gs leads to low foliar d13C values (Eq. (3)); all of which are consistent with the common ﬁndings with respect to gs in numerous studies (Chen, Bai & Han, 2002; Peri et al., 2012; Schifman et al., 2012). The decrease in the foliar d13C value between 2015 and 2016 at PBs and PCs along transects SKA and SKB, and at PB on the transect SBoydom are thus, also attributable to stomatal regulation of gas exchange between dry and wet conditions. Foliar δ13C values under sporadic waterlogging p gg g Willow leaves at the PAs of SKB and SBoydom in 2015 have the d13C values similar to or higher than those in their respective PB, even though PAs and PBs were situated under wet and dry conditions, respectively. The high d13C values in PAs can be explained by stomatal closure under waterlogging conditions. Decreasing gs was demonstrated under extremely wet conditions (Gomes & Kozlowski, 1980; Olivella et al., 2000; Copolovici & Niinemets, 2010; Li & Sugimoto, 2017). We suppose, as has been reported, that the ﬂooding reduces root hydraulic conductance and thereby, leaf water potential (Olivella et al., 2000; Islam & Macdonald, 2004), which in turn leads to decreased gs. Else et al. (2001) also suggested that stomatal closure upon waterlogging is caused by the production of abscisic acid, which may be related to the decrease of root hydraulic conductance and leaf water potential. Moreover, in the present study, we observed a rapid recovery in A but a slow recovery in gs after waterlogging ended (Figs. 6B and 6C). Thus, when the waterlogging was short and continual, low gs can contribute more to the foliar d13C values than A, resulting in the high foliar d13C values at SPh-PB, although the lack of statistical signiﬁcance (Fig. 6A). The co-occurrence of low gs with high foliar d13C values was also observed under waterlogging in ﬁeld trials (Ewe & Sternberg, 2003; Anderson et al., 2005) as well as in pot experiments (Li & Sugimoto, 2017). Therefore, we suggest that gs contributes more than A to the foliar d13C values under sporadic waterlogging caused by medium ﬂooding (“WL”; Fig. 3). The combined results of this and previous research indicates that increasing of the foliar d13C values (+0.8 ± 0.6‰) at SKA-PA between 2015 and 2016 were caused by stomatal closure in response to the waterlogging in 2016 (“WL”; Fig. 3). Very similar increases in the foliar d13C value were also observed in the SBoydom-PC, even though it was relatively distant from the river. The wetlands near SBoydom-PC may cause waterlogging similar to that experienced in the riverside sites (Fig. 3). Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Foliar δ13C values under continuous long period waterlogging The low d13C values, A, and gs under large ﬂooding were previously reported for a pot experiment involving the invasive wetland grass Phalaris arundinacea (Waring & Maricle, 2012). The aforementioned ﬁndings together suggest the hypothesis that long period waterlogging (or large ﬂooding) signiﬁcantly reduces the foliar d13C values compared to sporadic waterlogging (or small ﬂooding), and that the contribution of A and gs is highly dependent on the frequency and magnitude of waterlogging events. Thus, the large differences (Fig. 4) in hydrological conditions (i.e., “Dry,” “Wet,” “WL,” and “LWL”; Fig. 3) found in transects suggest that the possible foliar d13C values can correspond to scenario 3 in Fig. 1 (i.e., reduced foliar d13C values in long period waterlogging), which can be well interpreted by that both A and gs are affected by hydrological gradients (Fig. 7). on SKB and SBoydom decreased by 1.7 ± 0.6‰ and 2.1 ± 0.9‰, respectively, despite waterlogging occurring in both years. To date, very few studies have investigated the reasons for the lack of changes or negative shifts in foliar d13C value under waterlogging conditions. We propose that, under the long period waterlogging (“LWL”; Fig. 3) as caused by large ﬂooding observed at PAs on SKB and SBoydom in 2016, the changes in the A are also important factors controlling the foliar d13C values, besides gs. In the present study, low A was observed during submergence, before July 20 and more ﬂooded SPh-PA (Fig. 6C). It has been suggested that waterlogging induces low carboxylation rate by reducing the amount or activity of Rubisco enzyme (Vu & Yelenosky, 1992; Islam & Macdonald, 2004). According to Eq. (3), the foliar d13C values should be dependent on both A and gs; thus, low A may have caused the negative shifts in the foliar d13C value in LWL compared to WL, as the foliar d13C values at SPh-PA were slightly lighter than at SPh-PB, albeit insigniﬁcantly so (Fig. 6A). Therefore, the low foliar d13C values observed in the willows at the PAs on SKB and SBoydom in 2016, can be explained by this continuously low photosynthetic activity under long period waterlogging caused by large ﬂooding. The low d13C values, A, and gs under large ﬂooding were previously reported for a pot experiment involving the invasive wetland grass Phalaris arundinacea (Waring & Maricle, 2012). Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Foliar δ13C values under continuous long period waterlogging Sporadic waterlogging (“WL”; Fig. 3) related to medium ﬂooding increased the willow foliar d13C values. In 2016, however, the foliar d13C values at SKB-PA (waterlogging) were only slightly higher (approximately 0.4‰) than those at SKB-PB and SKB-PC. The foliar d13C values at SBoydom-PA (waterlogging) were even lower (approximately 1‰) than those at SBoydom-PB. Moreover, between 2015 and 2016 the foliar d13C values at PAs Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Figure 7 The possible foliar δ13C values with respect to physiological responses to various hydrological conditions. gs: stomatal conductance, A: photosynthesis activity. Dry and wet are with- out waterlogging, and WL and LWL represent waterlogging (continual) and long period waterlogging (continuous), respectively. Full-size  DOI: 10.7717/peerj.5374/ﬁg-7 Figure 7 The possible foliar δ13C values with respect to physiological responses to various hydrological conditions. gs: stomatal conductance, A: photosynthesis activity. Dry and wet are with- out waterlogging, and WL and LWL represent waterlogging (continual) and long period waterlogging (continuous), respectively. Full-size  DOI: 10.7717/peerj.5374/ﬁg-7 on SKB and SBoydom decreased by 1.7 ± 0.6‰ and 2.1 ± 0.9‰, respectively, despite waterlogging occurring in both years. To date, very few studies have investigated the reasons for the lack of changes or negative shifts in foliar d13C value under waterlogging conditions. We propose that, under the long period waterlogging (“LWL”; Fig. 3) as caused by large ﬂooding observed at PAs on SKB and SBoydom in 2016, the changes in the A are also important factors controlling the foliar d13C values, besides gs. In the present study, low A was observed during submergence, before July 20 and more ﬂooded SPh-PA (Fig. 6C). It has been suggested that waterlogging induces low carboxylation rate by reducing the amount or activity of Rubisco enzyme (Vu & Yelenosky, 1992; Islam & Macdonald, 2004). According to Eq. (3), the foliar d13C values should be dependent on both A and gs; thus, low A may have caused the negative shifts in the foliar d13C value in LWL compared to WL, as the foliar d13C values at SPh-PA were slightly lighter than at SPh-PB, albeit insigniﬁcantly so (Fig. 6A). Therefore, the low foliar d13C values observed in the willows at the PAs on SKB and SBoydom in 2016, can be explained by this continuously low photosynthetic activity under long period waterlogging caused by large ﬂooding. Foliar δ13C values under continuous long period waterlogging The aforementioned ﬁndings together suggest the hypothesis that long period waterlogging (or large ﬂooding) signiﬁcantly reduces the foliar d13C values compared to sporadic waterlogging (or small ﬂooding), and that the contribution of A and gs is highly dependent on the frequency and magnitude of waterlogging events. Thus, the large differences (Fig. 4) in hydrological conditions (i.e., “Dry,” “Wet,” “WL,” and “LWL”; Fig. 3) found in transects suggest that the possible foliar d13C values can correspond to scenario 3 in Fig. 1 (i.e., reduced foliar d13C values in long period waterlogging), which can be well interpreted by that both A and gs are affected by hydrological gradients (Fig. 7). on SKB and SBoydom decreased by 1.7 ± 0.6‰ and 2.1 ± 0.9‰, respectively, despite waterlogging occurring in both years. To date, very few studies have investigated the reasons for the lack of changes or negative shifts in foliar d13C value under waterlogging conditions. We propose that, under the long period waterlogging (“LWL”; Fig. 3) as caused by large ﬂooding observed at PAs on SKB and SBoydom in 2016, the changes in the A are also important factors controlling the foliar d13C values, besides gs. In the present study, low A was observed during submergence, before July 20 and more ﬂooded SPh-PA (Fig. 6C). It has been suggested that waterlogging induces low carboxylation rate by reducing the amount or activity of Rubisco enzyme (Vu & Yelenosky, 1992; Islam & Macdonald, 2004). According to Eq. (3), the foliar d13C values should be dependent on Thus, the large differences (Fig. 4) in hydrological conditions (i.e., “Dry,” “Wet,” “WL,” and “LWL”; Fig. 3) found in transects suggest that the possible foliar d13C values can correspond to scenario 3 in Fig. 1 (i.e., reduced foliar d13C values in long period waterlogging), which can be well interpreted by that both A and gs are affected by hydrological gradients (Fig. 7). Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 However, we note that scenario 3 in this study is a very simpliﬁed, schematic hypothesis, without quantitative meaning. There are other several potential factors for controlling the foliar d13C values, for example mesophyll conductance (gm) (Evans et al., 1986). As gs and gm were found tightly coupled (Vrábl et al., 2009), and both controlled the limitation of CO2 diffusion. Foliar δ13C values under continuous long period waterlogging Therefore, in our ﬁeld study of the determining factors of foliar d13C values, we mainly focused on gs which can be directly monitored. Detailed changes in the d13C value particularly between and within the four hydrological conditions will be illustrated in further studies with determination of these potential factors, including but not limited to gm changes under different water regimes, the quantitative meaning of gs and gm on foliar d13C values, and the features of gs and gm in species with different water tolerance. Spatial difference in the willow foliar δ13C value Linear Mixed Models analysis of the local scale random sampling indicated that only the willows in the dry larch forest were slightly but statistically enriched in 13C (F4,168 = 2.58, P = 0.039), compared to the other conditions, in 2016. It is accepted that the foliar d13C values are higher in dry than in wet conditions, due to stomatal regulation. In contrast, there was no statistical difference in the foliar d13C value for either year or among the hydrological conditions tested here (ﬂooding, ﬂooded, on land, and wetland) (Fig. 5). The ﬁrst three conditions were situated near the river and at different levels of ﬂooding, whereas the fourth was never affected by ﬂooding, although it was still abundant in water. These results are likely consistent with the lack of difference in the foliar d13C value among various hydrological conditions in mesic regions or periods, as reported previously (Garten & Taylor, 1992; Alstad et al., 1999). Nevertheless, relatively minor variations in the d13C value in willows growing under these hydrological conditions cannot be explained by the common dry-wet stomatal regulation theory mentioned above. On the other hand, our transect data in this study can explain why there was a slight variation in the foliar d13C value among random sampling sites in local scale in response to hydrological gradients as follows. In 2015, the water level in the river was low. Consequently, the hydrological status of the willows growing nearest the river in the “Flooded” zone, ranged from slight ﬂooding (similar to “Wet” in Figs. 3 and 4; i.e., “small ﬂooding” in Fig. 7) to continual ﬂooding (results in continual waterlogging, e.g., “WL” in Figs. 3 and 4; i.e., “medium ﬂooding” in Fig. 7). Slight ﬂooding near the wet condition zone caused the stomata to open and low foliar d13C values. In contrast, medium ﬂooding resulted in stomatal closure and high foliar d13C values. Therefore, under the “Flooded” condition, the d13C values varied between low and high. This behavior resembles the positive-negative shift in the foliar d13C value for the “On land” zone under dry-wet conditions. The same interpretation applies to the foliar d13C values measured in the “Wetland” (Fig. 5). p pp g In contrast, in 2016, the water level in the river was high. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Spatial difference in the willow foliar δ13C value Therefore, the hydrological status of the willows growing nearest the river in the “Flooding” zone varied between continual ﬂooding (results in continual waterlogging, e.g., “WL” in Figs. 3 and 4; i.e., “medium ﬂooding” in Fig. 7) and continuous ﬂooding (leads to long period waterlogging, e.g., “LWL” in Figs. 3 and 4; i.e., “large ﬂooding” in Fig. 7). Large ﬂooding Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 reduced both, A and foliar d13C values. As was the case for 2015, the conditions in the “Flooded” and “Wetland” zones of 2016 ranged between slight and continual waterlogging (similar to “Wet” and “WL,” respectively in Figs. 3 and 4), although only waterlogging in “Flooded” zones was caused by ﬂoods (i.e., “small ﬂooding” and “medium ﬂooding”; Fig. 7). Therefore, the foliar d13C values ranged between low and high in the “Flooding,” “Flooded,” and “Wetland” areas. These responses resemble the positive-negative shift in the foliar d13C value observed for “On land” under dry-wet conditions. In previous studies (Garten & Taylor, 1992; Alstad et al., 1999), very small differences in the foliar d13C value of plants growing near rivers were detected among the diverse hydrological conditions, where waterlogging frequently occurred. These minor differences can also be explained by the physiological responses of willows related to the different hydrological conditions (Fig. 7). If the d13C values in other organs correlate with those determined by the leaves, then historical records of the wide swings in hydrological conditions could be reconstructed using the d13C records, such as those obtained from tree ring cellulose. CONCLUSIONS To illustrate the effects of hydrological conditions on the d13C values in leaves, we measured the foliar d13C values of willows at three different points, along three transects near the Indigirka River, under several major hydrological conditions (Fig. 7). Under normal hydrological conditions, the foliar d13C values were lower under wet conditions (along rivers and/or during a wet year) than under dry conditions (far from the river and/or during a dry year), because the former conditions allowed for stomatal opening. On the other hand, under abnormal hydrological conditions, such as waterlogging, high foliar d13C values were found, because medium ﬂooding induced stomatal closure. Moreover, long period waterlogging decreased foliar d13C value by reducing photosynthetic activity. Thus, there was a small variation in the foliar d13C value (-31.6 to -25.7‰) in the Indigirka River lowland, despite large diversity in the hydrological conditions (Fig. 5). These results demonstrate that the foliar d13C values reﬂect hydrological conditions even in mesic environments (Fig. 7). If the foliar d13C values correlate with those in other organs and tissues (such as tree-ring cellulose), they can be used to reconstruct the hydrological and vegetation changes that have occurred in mesic regions. We suggest that further clarifying of the effects of waterlogging on the foliar or tree ring d13C values in conducting laboratory experiments under controlled conditions and in ﬁeld can be highly useful for better interpretation in the d13C values of plant products. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Competing Interests The authors declare that they have no competing interests. Author Contributions Rong Fan conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared ﬁgures and/or tables, authored or reviewed drafts of the paper, approved the ﬁnal draft. Tomoki Morozumi contributed reagents/materials/analysis tools, approved the ﬁnal draft, helped with sampling. Troﬁm C. Maximov approved the ﬁnal draft, supported the ﬁeld work. Atsuko Sugimoto conceived and designed the experiments, authored or reviewed drafts of the paper, approved the ﬁnal draft, supported the ﬁeld work. Funding This work was supported by the China Scholarship Council (No. 201406180095); Japan Science and Technology Agency (Belmont Forum, project COPERA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ACKNOWLEDGEMENTS We kindly thank Profs. A. Maximov, A. Kononov, and the other members of the IBPC and Ms. T. Stryukova and Mr. S. Ianygin of the Allikha Nature Protection Ofﬁce for supporting our ﬁeldwork near Chokurdakh, and Dr. S. Tei, Mr. R. Shingubara and S. Takano for their assistance in both ﬁeld and labwork. We also thank Mss. Y. Hoshino, S. Nunohashi, and H. Kudo for their supports in labwork, and Dr. L. Chen for his helpful advice in paper writing. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Grant Disclosures The following grant information was disclosed by the authors: China Scholarship Council: 201406180095. Japan Science and Technology Agency (Belmont Forum, project COPERA). Field Study Permissions The following information was supplied relating to ﬁeld study approvals (i.e., approving body and any reference numbers): Field experiments were approved by Institute for Biological Problems of Cryolithozone, Siberian Branch of Russian Academy of Science, Hokkaido University, and North-Eastern Federal University. Data Availability The following information was supplied regarding data availability: The raw data are provided in the Supplemental Files. The raw data are provided in the Supplemental Files. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Supplemental Information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.5374#supplemental-information. Alstad KP, Welker JM, Williams SA, Trlica MJ. 1999. 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https://openalex.org/W2322414352	https://europepmc.org/articles/pmc4814530?pdf=render	English	null	Multiclass Classification by Adaptive Network of Dendritic Neurons with Binary Synapses Using Structural Plasticity	Frontiers in neuroscience	2,016	cc-by	19,029	ORIGINAL RESEARCH published: 31 March 2016 doi: 10.3389/fnins.2016.00113 Multiclass Classiﬁcation by Adaptive Network of Dendritic Neurons with Binary Synapses Using Structural Plasticity School of Electrical and Electronic Engineering, Nanyang Technological University, Singapore, Singapore The development of power-efﬁcient neuromorphic devices presents the challenge of designing spike pattern classiﬁcation algorithms which can be implemented on low-precision hardware and can also achieve state-of-the-art performance. In our pursuit of meeting this challenge, we present a pattern classiﬁcation model which uses a sparse connection matrix and exploits the mechanism of nonlinear dendritic processing to achieve high classiﬁcation accuracy. A rate-based structural learning rule for multiclass classiﬁcation is proposed which modiﬁes a connectivity matrix of binary synaptic connections by choosing the best “k” out of “d” inputs to make connections on every dendritic branch (k << d). Because learning only modiﬁes connectivity, the model is well suited for implementation in neuromorphic systems using address-event representation (AER). We develop an ensemble method which combines several dendritic classiﬁers to achieve enhanced generalization over individual classiﬁers. We have two major ﬁndings: (1) Our results demonstrate that an ensemble created with classiﬁers comprising moderate number of dendrites performs better than both ensembles of perceptrons and of complex dendritic trees. (2) In order to determine the moderate number of dendrites required for a speciﬁc classiﬁcation problem, a two-step solution is proposed. First, an adaptive approach is proposed which scales the relative size of the dendritic trees of neurons for each class. It works by progressively adding dendrites with ﬁxed number of synapses to the network, thereby allocating synaptic resources as per the complexity of the given problem. As a second step, theoretical capacity calculations are used to convert each neuronal dendritic tree to its optimal topology where dendrites of each class are assigned different number of synapses. The performance of the model is evaluated on classiﬁcation of handwritten digits from the benchmark MNIST dataset and compared with other spike classiﬁers. We show that our system can achieve classiﬁcation accuracy within 1 −2% of other reported spike-based classiﬁers while using much less synaptic resources (only 7%) compared to that used by other methods. Further, an ensemble classiﬁer created with adaptively learned sizes can attain accuracy of 96.4% which is at par with the best reported performance of spike-based classiﬁers. Moreover, the proposed method achieves this by using about 20% of the synapses used by other spike algorithms. We also present results of applying our algorithm to classify the MNIST-DVS dataset collected from a real spike-based image sensor and show results Correspondence: Arindam Basu arindam.basu@ntu.edu.sg Correspondence: Arindam Basu arindam.basu@ntu.edu.sg Specialty section: This article was submitted to Neuromorphic Engineering, a section of the journal Frontiers in Neuroscience Received: 06 September 2015 Accepted: 07 March 2016 Published: 31 March 2016 Edited by: Themis Prodromakis, University of Southampton, UK Reviewed by: Omid Kavehei, Royal Melbourne Institute of Technology, Australia Hesham Mostafa, Institute for Neuroinformatics, Switzerland 1. INTRODUCTION area. For example, a spiking network classiﬁer implemented on a neuromorphic hardware system achieved performance comparable with standard machine learning linear classiﬁer and exhibited tolerance against variability by using population coding (Schmuker et al., 2014). However, a limitation of this model is the large number of high resolution weights used to attain the reported performance. Similarly, spike classiﬁers consisting of Restricted Boltzmann Machine (RBM) constructed with integrate and ﬁre neurons, use a large number of recurrent synaptic connections (Neftci et al., 2014; O’Connor et al., 2013) rendering these algorithms impractical for compact VLSI implementation. A simple and robust solution to the problem of device mismatch can be obtained by using binary synaptic weights. A spike-based STDP learning rule using bistable synapses was implemented in Brader et al. (2007) with the VLSI implementation in Mitra et al. (2009) to classify complex stimuli. A pool of neurons was used to improve the classiﬁcation accuracy by employing a voting scheme, which again leads to the problem of increased number of synapses. Digital implementations do not suﬀer from mismatch issues like their analog counterparts; however, the usage of a lot of memory to implement high resolution weights for deep networks increases chip area signiﬁcantly. There has been signiﬁcant research in the last decade aimed at designing neuromorphic systems which can emulate the architectural and computational principles of the brain. These systems exploit the spike-based operation of human brain, with minimal power consumption during long inactive periods, to implement power-eﬃcient neuromorphic devices. Moreover, this attempt to mimic the neuronal function can enable us to design event-driven, compact hardware systems which can provide eﬃcient, real-time, intelligent solutions for several applications like robotics and brain-machine interfaces. Conversely, the neuromorphic systems can be used to understand the working principles of brain. The development of event-driven sensors like the artiﬁcial retina (Lichtsteiner et al., 2008; Posch et al., 2011; Serrano-Gotarredona and Linares-Barranco, 2013) and cochlea (Liu et al., 2013), which produce continuous and asynchronous spikes encoding the sensory information, make it essential to interface these sensors with spike-based classiﬁer systems to enable the classiﬁcation of real-world complex stimuli. The spike classiﬁcation algorithms designed to this eﬀect can also attain large computational power of spiking neural networks (Maass and Schmitt, 1999). Citation: Hussain S and Basu A (2016) Multiclass Classiﬁcation by Adaptive Network of Dendritic Neurons with Binary Synapses Using Structural Plasticity. Front. Neurosci. 10:113. doi: 10.3389/fnins.2016.00113 March 2016 \| Volume 10 \| Article 113 Frontiers in Neuroscience \| www.frontiersin.org 1 Structural Learning with Binary Synapses Hussain and Basu comparable to the best reported ones (88.1% accuracy). For VLSI implementations, we show that the reduced synaptic memory can save upto 4X area compared to conventional crossbar topologies. Finally, we also present a biologically realistic spike-based version for calculating the correlations required by the structural learning rule and demonstrate the correspondence between the rate-based and spike-based methods of learning. Keywords: active dendrite, structural plasticity, binary synapses, multiclass classiﬁcation, neuromorp 1. INTRODUCTION The spike-based neuromorphic systems implemented in very-large-scale integration (VLSI) technology consist of hybrid analog-digital circuits, where the neuronal and synaptic computations are usually performed in analog form on the chip (though TrueNorth Merolla et al., 2014 and Spinnaker Painkras et al., 2013 are notable exceptions) while the synaptic connectivity information is stored on or oﬀ-chip in a digital memory. An asynchronous communication protocol called the address-event representation (AER; Boahen, 2000; Choi et al., 2005; Serrano-Gotarredona et al., 2009; Vogelstein et al., 2007), is used to transmit neuronal spikes between neuromorphic chips on a shared fast digital bus. The AER-based neuromorphic systems have the added advantage of reconﬁgurability since the conﬁguration details of a network are stored in a separate memory, thereby giving the user ﬂexibility to reconﬁgure the network connectivity. A spike classiﬁcation model proposed in Hussain et al. (2013, 2015) oﬀers a solution to the problem of large number of synaptic weights by using a structural plasticity based learning rule which involves formation of sparse connections with binary weights. Moreover, it was shown that a simple correlation-based learning rule provides an alternative to the traditional weight- based learning rules and is more suitable for implementation on neuromorphic chips. The problem of reduced memory capacity of a network with binary synapses as compared with that of continuous-valued synaptic weights (Senn and Fusi, 2005) is alleviated by the use of nonlinear dendritic processing, which emerges due to the presence of voltage gated ion channels (London and Hausser, 2005; Magee, 2000). A limitation of this method is that it uses a preassigned network size and the number of dendrites and synapses required for solving a classiﬁcation problem of given complexity is not known. Hence, it is desirable to use an approach which learns to allocate the required number of synaptic resources for a speciﬁc problem. However, the statistical variations in VLSI devices which reduce the accuracy of the synaptic weights are a major cause for concern in attaining performance comparable to software simulations. To mitigate the eﬀect of increasing device mismatch with progressively shrinking transistor sizes, the usual solutions are to increase device sizes or employ a large number of neurons, both of which increase chip Several adaptive approaches have been used to control the network size and structure. Frontiers in Neuroscience \| www.frontiersin.org 2. MATERIALS AND METHODS The unique contributions of this work are: (1) Showing that for ensembles of NNLD with the same number of synapses, having a single dendritic branch (perceptron) or having too many dendrites are sub-optimal; the optimal cases is a moderate size of the dendritic tree. (2) Developing an algorithm that adapts the size of the dendritic tree for each class according to the diﬃculty of classifying that pattern category. (3) Applying this network to the problem of handwritten digit recognition task from MNIST and MNIST-DVS datasets to show its beneﬁt in achieving high accuracy with very small memory usage for weights. (4) Demonstrating memory size reductions possible in VLSI implementations by using this training method. d X i=1 wij = k for j = 1, · · · , m (1) (1) This model was used to perform supervised binary classiﬁcation of spike patterns. In our current work, we extend the model to perform multiclass classiﬁcation of spike patterns belonging to NC classes. The multiclass classiﬁer consists of (+) and (−) neurons corresponding to all NC classes. The inputs to the neurons for class µ are received from a pair of excitatory and inhibitory dendritic trees, also referred to as positive and negative dendritic trees (PDT and NDT), respectively, each with m dendrites. These input currents for class µ neurons are given by: This model was used to perform supervised binary classiﬁcation of spike patterns. In our current work, we extend the model to perform multiclass classiﬁcation of spike patterns belonging to NC classes. The multiclass classiﬁer consists of (+) and (−) neurons corresponding to all NC classes. The inputs to the neurons for class µ are received from a pair of excitatory and inhibitory dendritic trees, also referred to as positive and negative dendritic trees (PDT and NDT), respectively, each with m dendrites. These input currents for class µ neurons are given by: Iµ+ in (t) = Iµ PDT(t) −Iµ NDT(t) (2) Iµ− in (t) = Iµ NDT(t) −Iµ PDT(t) (3) (2) (2) (3) (3) The paper is organized as follows. First, the rate-based multiclass spike pattern classiﬁcation model is presented in Section 2.1.1, followed by the description of an ensemble method which combines the outputs of several dendritic classiﬁers to obtain improved classiﬁcation accuracy in Section 2.1.2. 2. MATERIALS AND METHODS adding further hidden units and weights to implement the desired mapping (Islam et al., 2009; Kwok and Yeung, 1997; Lahnajarvi et al., 2002). The second approach for automating the design of appropriate neural network is by pruning in which a network larger than necessary is trained and then redundant connections and/or neurons are removed until an acceptable solution is obtained. A group of pruning algorithms eliminate a neuron or a connection which have the least eﬀect on the error function (Karnin, 1990). The other group of pruning algorithms are referred to as regularization methods which add a penalty term proportional to the sum of weights to the objective function. Hence, the unnecessary weights are driven to zero during training and are eliminated in eﬀect (Kwok and Yeung, 1996). Several other pruning methods are reviewed in Islam et al. (2009) and Reed (1993). Another class of algorithms that are developed to control the network size and structure use a hybrid approach of combining the constructive and pruning methods (Fiesler, 1994). The growth and pruning algorithms discussed here have not considered the number of dendrites as an adaptive parameter. We have proposed a margin-based neuron model with nonlinear dendrites (NLD) for spike pattern classiﬁcation (Hussain et al., 2015). The model comprises nonlinear neurons having lumped dendritic nonlinearity where a nonlinear neuron (NL-neuron) consists of multiple (m) dendritic branches with each branch governed by its nonlinearity b() and k excitatory synapses on each branch driven by one of the d input components (Figure 1A). The model uses binary weight for the ith synapse on the jth dendrite, wij ∈{0, 1}, where i ∈ 1, · · · d , j ∈{1, · · · m}. The advantage of such binary weight connections are in hardware implementations since they require much less memory resources, are more resistant to mismatch in analog implementations and also enable easy digital implementations as done in Merolla et al. (2014). Moreover, we enforce sparse connectivity on each dendritic branch (k << d) and allow the learning to choose the best “k” connections on each branch. Hence, we can write: In this paper, we present a multiclass classiﬁer using neurons with nonlinear dendrites (NNLD) and a structural learning rule for ﬁnding sparse, binary weight matrices. 1. INTRODUCTION A constructive approach involves training with a minimal architecture, for example a single hidden-layered network with one hidden neuron, and then March 2016 \| Volume 10 \| Article 113 2 Structural Learning with Binary Synapses Hussain and Basu Frontiers in Neuroscience \| www.frontiersin.org March 2016 \| Volume 10 \| Article 113 2. MATERIALS AND METHODS In Section 2.1.3, we propose an adaptive structural learning scheme which involves growth of the network by adding dendrites based on the progress of learning process. The performance of diﬀerent learning schemes on classiﬁcation of handwritten digit samples is demonstrated in Sections 3.2–3.4, while noise sensitivity analysis is in Section 3.5 and analysis of dendrite weights in Section 3.6. Next, in Section 3.7, we present an approach to optimize the performance of our method by utilizing theoretically determined optimal neuron topology. Finally, the evaluation of our algorithm on event-based MNIST-DVS dataset is presented in Section 3.8 followed by a comparison of the performance of our model with the results obtained using other spike-based classiﬁcation algorithms in Section 3.9. The relevance of our work in terms of the biological plausibility, comparisons with other related studies, hardware considerations and a discussion of future work is included in Section 4. where µ ∈{1, · · · NC} and Iµ PDT(t), Iµ NDT(t) are the currents generated by the PDT and NDT of class µ, calculated by taking the sum of dendritic output currents Iµ PD,j(t) and Iµ ND,j(t) respectively, which can be expressed as nonlinear functions of the total synaptic current on a dendrite. To make notations simpler for the ease of reading, we drop the superscript µ for the µth class in the rest of the paper except where the outputs of two or more classes are compared. Hence, we denote the PDT/NDT currents for class µ neuron as IDT(t) and the output current of the jth dendrite of PDT/NDT as ID,j(t). Therefore, the equations for IDT(t) can be written as: IDT(t) = X j ID,j(t) (4) = X j b  X i wij  X tij<t K(t −tij)     (5) = X j b(zj(t)) (6) (4) (5) (6) March 2016 \| Volume 10 \| Article 113 March 2016 \| Volume 10 \| Article 113 Frontiers in Neuroscience \| www.frontiersin.org 3 Structural Learning with Binary Synapses Hussain and Basu Here K(t) denotes the postsynaptic current (PSC) generated by the spike input at a synapse of the PDT or NDT of class µ and is given by Gutig and Sompolinsky (2006): Here K(t) denotes the postsynaptic current (PSC) generated by the spike input at a synapse of the PDT or NDT of class µ and is given by Gutig and Sompolinsky (2006): with NLDs. 2.1.1. Rate-Based Learning Scheme for Multiclass Classiﬁcation The rate-based learning rule for NLD model is valid for rate encoded inputs like Poisson spike trains and place/synchrony encoded single spike patterns. This validity results from the fact that for such spike inputs, the average synaptic activation zsyn,ij = 1 T R T 0 P tij K(t −tij)dt is directly proportional to the input arriving at that synapse, xij, where T is the pattern duration (Hussain et al., 2015). This reduced model was developed to improve the training time and it uses encoded binary vectors obtained by mapping spike train with “high” ﬁring rate or single spike to binary value “1” and spike train with “low” ﬁring rate or absence of spike to binary value “0.” A rate-based NLD model for multiclass classiﬁcation was proposed in Hussain et al. (2014), which consisted of NC neurons representing NC classes. Here, we present a modiﬁed version of this model consisting of PDT and NDT corresponding to each of the NC classes. The output of the PDT and NDT of class µ to input x is given by: The dynamics of the membrane potential Vm(t) of the (+) and (−) neurons of class µ is explained through the equations given below: τV dVm dt = (u −Vm) + Iin(t) (9) τu du dt = −u (10) If Vm ≥Vthr, Vm →Vreset; u →ureset ureset = Vreset < 0 nspk →nspk + 1 (9) (10) where u denotes a hyperpolarization variable which relaxes back to 0 with a time constant τu and is set to ureset after a postsynaptic spike and τV and τu are the time constants governing the fast and slow dynamics of the membrane voltage and hyperpolarization respectively. Here, the variables Vm(t), u and nspk are used to describe the dynamics of both (+) and (−) neurons of class µ and the input current Iin(t) can be computed using Equations (2) and (3). 2. MATERIALS AND METHODS For these schemes using binary synapses, we present structural learning rules, which involve the modiﬁcation of connection matrix instead of connection weights. The motivation for using rate-based learning is driven by the fact that the spike- time-based learning requires signiﬁcant processing time and therefore, to mitigate this problem we have used the strategy of training on mean rate inputs to reduce the training time. With the use of faster rate-based learning, we further propose an ensemble method which combines the outputs of several NLD classiﬁers in order to achieve performance gain over individual classiﬁers. We also develop an adaptive learning scheme which learns to allocate the required number of dendrites to a neural network along with learning the connection patterns on these dendrites. The bio-realistic spike-based approximation of the structural learning rule for multiclass classiﬁcation is an extension of an online spike-based binary classiﬁcation rule proposed in our earlier work (Hussain et al., 2015). The derivation of this rule and the correspondence between our spike-based and rate-based learning schemes are presented in the Appendix. Next, we describe the rate-based learning rule. K(t −tij) = I0 exp[−(t −tij)/τf ] −exp[−(t −tij)/τr] (7) (7) where tij denotes the times at which spikes arrive at this synapse; I0 is the normalization constant; and τf and τr are the fall and rise time constants of the PSC respectively; zj(t) is the total synaptic activation on the jth dendrite and b(zj(t)) is the dendritic nonlinear function given by: b(zj(t)) = g(zj(t) −zleak,j(t))(zj(t) −zleak,j(t))2 (8) (8) Here, g() is a Heaviside step function that gives an output 1 at all times where the argument (zj(t) −zleak,j(t)) is positive and 0 otherwise and zleak,j(t) is the average synaptic activation on the jth branch corresponding to the initial random connections. The use of zleak,j(t) term serves to balance the excitation on each branch by subtracting the mean activation level on that branch, which can be regarded as a signal from a pool of inhibitory neurons. Finally, the spike output nspk of a neuron of class µ, receiving the input current Iin(t), is generated using the leaky integrate and ﬁre neuron model described next. Frontiers in Neuroscience \| www.frontiersin.org 2.1.1. Rate-Based Learning Scheme for Multiclass Classiﬁcation g (5) Steps (1)–(4) are repeated until either all the patterns have been memorized or nmin (= 150) local minima are encountered. At this point, the learning process is stopped. The connection patterns corresponding to the best minimum become the ﬁnal learned synaptic connections of the neuron. The learning process which is based on the mechanism of structural plasticity involves formation and elimination of synaptic connections. The connections are modiﬁed by computing a metric based on correlation values cij for each synapse of the PDT and NDT. Hence, the learning rule for the rate-based learning scheme is given by: After the completion of training, the ﬁnal learned connections are saved and then used to calculate the error rate on the spike test pattern set for the testing phase. This is done by mapping the learned connections onto an equivalent spiking network by introducing synaptic integration and spike initiation mechanisms. The testing classiﬁcation output is computed using Equation (11). For class µ, for PDT: cPDT ij =< xPDT ij bPDT j sgn(yd µ −yµ) > (17) For class µ, for NDT: cNDT ij = −< xNDT ij bNDT j sgn(yd µ −yµ) > (18) (17) 2.1.1. Rate-Based Learning Scheme for Multiclass Classiﬁcation aPDT(x) = m X j=1 b   d X i=1 wPDT,ijxPDT,ij   (12) aNDT(x) = m X j=1 b   d X i=1 wNDT,ijxNDT,ij   (13) (12) (13) As shown in Figure 1B, the outputs of all the (+) and (−) neurons are connected to a WTA circuit to generate the overall classiﬁer output as: The diﬀerence of outputs of the positive and negative dendritic trees of each class, o(x) = (aPDT(x) −aNDT(x)), are then used as inputs to the WTA circuit which computes the overall output and therefore decision of the classiﬁcation task, y(x) using a similar logic as in Equation (11). yµ = g(oµ spk −oν spk), oν spk ≥oξ spk, ∀ν, ξ ̸= µ (11) (11) where µ, ν, ξ are integers in the range [1, NC] and yµ is the binary-valued µth component of the NC-dimensional output y, oµ spk = nµ+ spk −nµ− spk ; nµ+ spk and nµ− spk are the spike counts of the (+) and (−) neurons of class µ respectively. yµ(x) = g(oµ(x) −oν(x)), oν(x) ≥oξ(x), ∀ν, ξ ̸= µ (14) (14) During training, the classiﬁer output is computed using a margin- based output function, gmargin(x) instead of g(x) to enforce a In this paper, we have developed rate-based and spike-time- based learning schemes for our multiclass classiﬁcation model March 2016 \| Volume 10 \| Article 113 Frontiers in Neuroscience \| www.frontiersin.org 4 Structural Learning with Binary Synapses Hussain and Basu margin around the classiﬁcation boundary. The g(x) function is only used during the testing phase to calculate the output. Therefore, the classiﬁer output during training calculated using the gmargin(x) function is given by: margin around the classiﬁcation boundary. The g(x) function is only used during the testing phase to calculate the output. Therefore, the classiﬁer output during training calculated using the gmargin(x) function is given by: At the start of the learning process, the input connections for all the PDT and NDT corresponding to NC classes each with m dendritic branches, k synaptic contacts per branch and s = m×k total synapses are initialized by randomly selecting aﬀerents from among d input lines with weight wij = 1. Training set consisting of P input patterns (x) belonging to NC classes is presented. Frontiers in Neuroscience \| www.frontiersin.org 2.1.1. Rate-Based Learning Scheme for Multiclass Classiﬁcation The connection matrix corresponding to the local minimum that gave the least error is saved. (4) Synaptic connections are modiﬁed if the replacement led to either a reduction or no change in error rate. If the error increased with the replacement, a new replacement set R is created. If the error does not decrease after repeating this step nch (= 50) times, we assume a local minimum is encountered. We then do a replacement with the last choice of R, even if it increases error in an attempt to escape the local minimum. The connection matrix corresponding to the local minimum that gave the least error is saved. (4) Synaptic connections are modiﬁed if the replacement led to either a reduction or no change in error rate. If the error increased with the replacement, a new replacement set R is created. If the error does not decrease after repeating this step nch (= 50) times, we assume a local minimum is encountered. We then do a replacement with the last choice of R, even if it increases error in an attempt to escape the local minimum. The connection matrix corresponding to the local minimum that gave the least error is saved. (2) Cross validation set patterns are presented and the values αµν = (oν(x) −oµ(x)) are recorded for all the cases for which patterns from class µ are misclassiﬁed as belonging to class ν. (3) The set of αµν values for each class µ ∈{1, · · · NC} are saved. The margin for class µ, δµ is set to the highest value of αµν. (4) δµ value is reduced to 80% of its present value whenever learning algorithm gets stuck in the same local minimum for 5 consecutive times. (3) The set of αµν values for each class µ ∈{1, · · · NC} are saved. The margin for class µ, δµ is set to the highest value of αµν. g g µ (4) δµ value is reduced to 80% of its present value whenever learning algorithm gets stuck in the same local minimum for 5 consecutive times. g (5) Steps (1)–(4) are repeated until either all the patterns have been memorized or nmin (= 150) local minima are encountered. At this point, the learning process is stopped. The connection patterns corresponding to the best minimum become the ﬁnal learned synaptic connections of the neuron. 2.1.1. Rate-Based Learning Scheme for Multiclass Classiﬁcation The learning process comprises the following steps in every iteration consisting of the presentation of all P patterns: yµ(x) = gmargin(oµ(x) −oν(x)), oν(x) ≥oξ(x) yν(x) = gmargin(oν(x) −oµ(x)), ∀ν, ξ ̸= µ (1 (15) The function gmargin() is deﬁned as: The function gmargin() is deﬁned as: (1) The misclassiﬁcation error rate is calculated by taking the average of the fraction of patterns for which yµ ̸= yd µ for any µ ∈{1, · · · NC}. gmargin(α) = 1 if α ≥δ = 0 if α ≤−δ = 0.5 δ α + 0.5 otherwise (16) (2) For each dendritic tree of class µ, a random set T consisting of nT (< s) synapses is selected as candidates for replacement. (2) For each dendritic tree of class µ, a random set T consisting of nT (< s) synapses is selected as candidates for replacement. (16) (3) The synapse with the lowest cij in T is targeted for replacement and the dendrite jT on which it is located is identiﬁed. A random replacement set R is created by placing nR “silent” synapses from d input lines (nR < d) on the jth T dendrite. The synapse with the lowest cij in T is replaced by the best-performing (maximum cij) synapse in R. The silent synapses do not contribute to the calculation in step (1). where the margin is set using diﬀerent values of the parameter δ for diﬀerent classes to which input patterns belong. The value of margin δ for each class is determined using the following steps: (1) Multi-class model is ﬁrst trained using the g(x) function, where the output of the model is calculated using 14. Connection matrices for all neurons are saved. (1) Multi-class model is ﬁrst trained using the g(x) function, where the output of the model is calculated using 14. Connection matrices for all neurons are saved. (4) Synaptic connections are modiﬁed if the replacement led to either a reduction or no change in error rate. If the error increased with the replacement, a new replacement set R is created. If the error does not decrease after repeating this step nch (= 50) times, we assume a local minimum is encountered. We then do a replacement with the last choice of R, even if it increases error in an attempt to escape the local minimum. 2.1.2. Ensemble Learning Scheme for Multiclass Classiﬁcation where the desired output yd is available as the teacher signal during the training phase and is a NC-dimensional binary vector consisting of (NC −1) zeros and a 1 corresponding to the class to which the input pattern belongs; bj is the output of the jth dendrite of a PDT or NDT; sgn() is the signum function with a value of 1 for yd µ > yµ, −1 for yd µ < yµ and 0 for yd µ = yµ; and the output y is computed using Equation (15). We have also used an ensemble method in this work, where several classiﬁers when combined together yield better classiﬁcation accuracy than any of the single classiﬁers in the ensemble. Our ensemble model consists of individually trained NLD classiﬁers which are then combined to classify novel test patterns. Since, previous research has demonstrated that combining identical classiﬁers doesn’t produce any gain over individual classiﬁers (Opitz and Maclin, 1999), hence we created ensemble by combining several classiﬁers which are individually trained and disagree on their predictions. The complementary information about the novel patterns obtained from diﬀerent classiﬁers can be exploited to produce a more The connection changes are done by using the following logic: a synapse with the smallest cij value corresponds to a poorly- performing synapse and is a candidate for replacement. To replace this synapse, a set of silent synapses are ﬁrst formed on the chosen branch as possible candidates for the new connection. The silent synapse with the highest cij is chosen as the replacement. March 2016 \| Volume 10 \| Article 113 5 Structural Learning with Binary Synapses Hussain and Basu FIGURE 1 \| (A) A neuron consisting of nonlinear dendrites characterized by the lumped nonlinear function b() for every dendritic branch. It forms k binary connections from d input afferents on m dendrites. (B) Architecture of a multiclass pattern classiﬁer consisting of nonlinear neurons receiving inputs from PDT and NDT and the neurons connected to a WTA to compare the outputs of the NC classes. The output of the classiﬁer is equal to 1 for the class corresponding to the highest output oµ. FIGURE 1 \| (A) A neuron consisting of nonlinear dendrites characterized by the lumped nonlinear function b() for every dendritic branch. It forms k binary connections from d input afferents on m dendrites. 2.1.2. Ensemble Learning Scheme for Multiclass Classiﬁcation To address this problem, we have developed an adaptive learning rule for allocating the number of dendrites according to the diﬃculty of the problem being solved as well as the diﬃculty to learn a speciﬁc class. This scheme is discussed next. The performance of ensemble model gives us an insight into the number of component classiﬁers needed in an ensemble. However, it is impractical to use a large number of classiﬁers in an ensemble due to the long training time required and also because it leads to increased synaptic resources. Therefore, we use the ensemble created with a few classiﬁers as a trade- oﬀbetween trainability and accuracy. Moreover, it is not clear what level of complexity is required for individual classiﬁers and whether an ensemble of perceptrons will perform better than that of complex dendritic trees. To address this problem, we have developed an adaptive learning rule for allocating the number of dendrites according to the diﬃculty of the problem being solved as well as the diﬃculty to learn a speciﬁc class. This scheme is discussed next. FIGURE 2 \| Ensemble classiﬁer combining the intermediate class-speciﬁc outputs oµ n to compute the combined class outputs Oµ, which are compared by the WTA to generate the ﬁnal classiﬁer output ˆy. trees in the model. Since, it is diﬃcult to choose an optimal value of m that matches the complexity of a given problem, we have proposed an algorithm in which the number of dendrites corresponding to each class is learned during the training process. The value of m is adapted for each class dendritic trees independently while the number of synapses per branch, k, is kept 2.1.2. Ensemble Learning Scheme for Multiclass Classiﬁcation (B) Architecture of a multiclass pattern classiﬁer consisting of nonlinear neurons receiving inputs from PDT and NDT and the neurons connected to a WTA to compare the outputs of the NC classes. The output of the classiﬁer is equal to 1 for the class corresponding to the highest output oµ. FIGURE 2 \| Ensemble classiﬁer combining the intermediate class-speciﬁc outputs oµ n to compute the combined class outputs Oµ, which are compared by the WTA to generate the ﬁnal classiﬁer output ˆy. accurate overall output. In order to generate diﬀerent predictions for diﬀerent classiﬁers, individual networks were initialized with diﬀerent random synaptic connections. Figure 2 shows the basic framework for the classiﬁer ensemble scheme. It consists of N individually trained multiclass classiﬁers as members of the ensemble. Each classiﬁer generates the intermediate output, oµ n (x), which is the diﬀerence in outputs of PDT and NDT for class µ of the nth classiﬁer. The intermediate outputs are combined in a class-speciﬁc manner to give Oµ(x) = PN n=1 oµ n (x). Finally, the ensemble output is generated using: accurate overall output. In order to generate diﬀerent predictions for diﬀerent classiﬁers, individual networks were initialized with diﬀerent random synaptic connections. Figure 2 shows the basic framework for the classiﬁer ensemble scheme. It consists of N individually trained multiclass classiﬁers as members of the ensemble. Each classiﬁer generates the intermediate output, oµ n (x), which is the diﬀerence in outputs of PDT and NDT for class µ of the nth classiﬁer. The intermediate outputs are combined in a class-speciﬁc manner to give Oµ(x) = PN n=1 oµ n (x). Finally, the ensemble output is generated using: ˆyµ(x) = g Oµ(x) −Oν(x) , Oν(x) ≥Oξ(x), ∀ν, ξ ̸= µ (19) The performance of ensemble model gives us an insight into the number of component classiﬁers needed in an ensemble. However, it is impractical to use a large number of classiﬁers in an ensemble due to the long training time required and also because it leads to increased synaptic resources. Therefore, we use the ensemble created with a few classiﬁers as a trade- oﬀbetween trainability and accuracy. Moreover, it is not clear what level of complexity is required for individual classiﬁers and whether an ensemble of perceptrons will perform better than that of complex dendritic trees. Frontiers in Neuroscience \| www.frontiersin.org 2.1.3. Adaptive Learning Scheme for Multiclass Classiﬁcation (2) This is done by drawing k random connections from the d input lines to represent a new dendrite. The connection matrix is updated by appending the new dendrite to the existing matrix as a new row. The value of m for class µ is incremented according to: CNL = 2log2 f + m −1 m (21) (21) mPDT →mPDT + 1 mNDT →mNDT + 1 (20) mPDT →mPDT + 1 mNDT →mNDT + 1 (20) (20) where f = k + d −1 k is the number of distinct branch where f = k + d −1 k where f = k + d −1 k is the number of distinct branch (3) After adding a dendrite, error rate for cross validation set is computed. If this validation error increases in the last 3 dendrite addition steps, the learning process is stopped. The connections are frozen and saved for the testing phase. (3) After adding a dendrite, error rate for cross validation set is computed. If this validation error increases in the last 3 dendrite addition steps, the learning process is stopped. The connections are frozen and saved for the testing phase. functions. As shown in the inset plot for the optimal topology determination step in Figure 3, CNL plotted as a function of m has a maximum at mopt for a ﬁxed value of sopt. These derivations were also discussed in our previous work (Hussain et al., 2015). We can now use this theory to change the structure of the µth neuron to optimize capacity while preserving the same number of synapses. Hence, by setting sµ opt = sµ adapt (ﬁxing the area of rectangle as shown in step-2 in Figure 3), the theoretical optimal topology (mµ opt, kµ opt) is determined such that sµ opt = sµ adapt = mµ opt × kµ opt. The use of this approach to boost the capacity of our adaptively learned dendritic network is demonstrated in Section 3.7. The above steps are added after the step (4) of the rate-based learning algorithm in Section 2.1.1 as steps 4.1, 4.2, and 4.3. All the steps from (1)–(4.3) are then repeated until learning stops by one of the stopping conditions discussed in step (5). 2.1.3. Adaptive Learning Scheme for Multiclass Classiﬁcation For the learning schemes described above, a ﬁxed number of dendrites (m) was chosen and assigned to all the dendritic March 2016 \| Volume 10 \| Article 113 Frontiers in Neuroscience \| www.frontiersin.org 6 Structural Learning with Binary Synapses Hussain and Basu all neurons and just vary the number of dendrites per neuron, m. Hence, at the end of the adaptation, both PDT and NDT of the µth class have a total number of synapses sµ adapt given by sµ adapt = mµ adapt × k, where mµ adapt is the number of dendritic branches learned for the µth class neurons after adaptation. This is shown as the step-1 in Figure 3, where the topology of a class neuron is denoted by the rectangle with its sides representing m and k values. constant throughout. Hence, the learning process generates the relative sizes of the connection matrix for each neuron as well as the optimal sparse connections within that matrix. The learning rule used is same as that for the rate-based scheme with ﬁxed m except that all the computations in every iteration are done for the current value of m for each neuron. Additional steps are included in the learning algorithm to adapt the value of m. After making a correlation-based connection change, we check if the learning process for a particular class µ has slowed down using the following steps: However, from theoretical considerations of function counting as shown in Poirazi and Mel (2001), the theoretical capacity of a neuron with nonlinear dendrites with a ﬁxed value of total number of synapses (sopt) is maximum for a relatively large value of mopt and small value of kopt. This theoretical capacity was given by the combinatorial expression derived by counting all possible ways in which d aﬀerents can connect to synapses on dendrites resulting in distinct memory ﬁelds (Poirazi and Mel, 2001). Hence, for a neuron with NLDs, the capacity CNL in bits was calculated as: (1) If the error rate for class µ, which is calculated as the average of the number of patterns for which yµ ̸= yd µ, does not decrease after nch (= 50) iterations, the learning process for class µ has encountered a local minimum. In this case, a new dendrite is added to both PDT and NDT corresponding to class µ, if the error is the highest amongst all the NC classes. 3.1. Input Generation However, due to the long simulation times of the spike-based learning, we have used rate-based learning for the remaining analyses in the paper. The binary input vectors are used for rate-based training. For spike-based training, patterns of single spikes are used, which are generated by mapping binary input “xi = 1” to a single spike arriving at Tsyn = 100 ms and “xi = 0” to no spike, where the stimulus duration T = 200 ms. Testing for both forms of rate-based and spike-based learning is done on two types of spike inputs: 1) place/synchrony code of single spikes with jittered spikes arriving within a time window [Tsyn−1 2 , Tsyn+ 1 2 ] corresponding to binary input “1” and no spike for “0” input and diﬀerent amounts of jitter 1; and 2) rate encoded Poisson spike trains with mean ﬁring rates of fhigh and flow mapped to binary inputs “1” and “0” respectively. The Poisson spike inputs have only been used to test the noise sensitivity of the model. The parameters used for the rate-based and spike-based models are given in Tables 1, 2 respectively, unless stated otherwise. 3.3. Performance of the Ensemble Method Next, we studied the eﬀect of combining several NLD multiclass classiﬁers in an ensemble. These individual classiﬁers were trained on P = 5000 patterns using rate-based learning rule and tested on 10, 000 jittered single spike inputs with 1 = 10 ms. The network consisted of m = 2, 5, 8, 15, and 20 dendrites in PDT and NDT for each of NC = 10 classes. Hence, the total number of dendrites used in the ensemble is given by M = 2×m×NC ×N. As shown in Figure 5A, the error rate for m = 2 reduces by about 48% when up to 25 classiﬁers are added in the ensemble. However, the error rate doesn’t change by much or it increases slightly if further classiﬁers are combined. Moreover, most of the error reduction, 40% out of the total change of 48%, is achieved when ﬁrst 5 classiﬁers are added. We have also looked at the eﬀect of size of individual networks in the ensemble. Figure 5B illustrates this eﬀect where error rate is plotted as a function of the total number of dendrites in the ensemble, M. As shown, the error rate reduces with the number of classiﬁers for all values of m. 2.1.3. Adaptive Learning Scheme for Multiclass Classiﬁcation As shown in Figure 4, for the rate-based learning, the training error increases while the testing error on spike versions of the inputs reduces as the training set size increases. It is clear from this result that as more data is added, it becomes diﬃcult to memorize the data. However, the generalization performance improves with more training data. A comparison between the spike-based and rate-based learning schemes is included in the Appendix, where we have shown that we can achieve similar performance for these two forms of learning. However, due to the long simulation times of the spike-based learning, we have used rate-based learning for the remaining analyses in the paper. TABLE 1 \| Parameters for rate-based multiclass model. d m k nR nT Tsyn 784 10 10 25 25 100 ms 3. RESULTS TABLE 1 \| Parameters for rate-based multiclass model. TABLE 1 \| Parameters for rate-based multiclass model. d m k nR nT 784 10 10 25 25 3.1. Input Generation For a ﬁxed value of M, m = 2 gives the highest error rate, which reduces with larger values of m. However, as m is increased beyond a certain value (m = 8 in this case, the reduction in errors is not signiﬁcant, and therefore the use of more complex dendritic trees is not leading to signiﬁcant advantage in terms of performance. The signiﬁcance of using these spike inputs can be understood by considering that single spike representations are commonly used in time-to-ﬁrst-spike (TTFS) imagers (Chen and Bermak, 2007; Qi et al., 2004). These imagers when presented with binary images such as in the MNIST dataset used in this work, generate a cluster of spikes corresponding to the white pixels and another cluster much later in time corresponding to the black pixels. Such place/synchrony code is also abundantly used in neuroscience (Gerstner and Kistler, 2002). Moreover, rate encoded outputs are commonly available from neuromorphic sensors such as the artiﬁcial cochlea in Chan et al. (2007). Such rate encoded Poisson spike trains are also often used to test the performance of neuromorphic classiﬁers as demonstrated in Marti et al. (2015), O’Connor et al. (2013), and Brader et al. (2007). 3.1. Input Generation We have demonstrated the performance of our multiclass classiﬁcation NLD model on the MNIST dataset consisting of grayscale images of handwritten digits belonging to one of the NC = 10 classes (0 to 9). It is a benchmark machine learning dataset used previously to test the performance of many classiﬁcation algorithms (LeCun et al., 1998). The input patterns consisting of 28 × 28 images were converted into d = 784 dimensional binary vectors by thresholding. The training set consists of 20, 000 patterns randomly selected from the full MNIST dataset, with equal number of samples of each digit. The testing set consists of a total of 10, 000 patterns. In case of adaptive learning scheme, 20% of the training patterns comprise the cross validation set which is used to compute the error whenever a dendrite is added to the network and ﬁnally to stop adding dendrites when the cross validation error doesn’t reduce further. The learning procedure is stopped when either all the patterns are correctly classiﬁed or when 150 minima are encountered. For diﬀerent training runs 150 minima are encountered in diﬀerent number of learning iterations and for each data point generated, this training process is repeated 3 times. on P = 200 −20, 000 patterns using the rate-based learning scheme. Each training accuracy is obtained by averaging over 3 trials and the testing accuracy is computed by averaging across 10 presentations of 10, 000 test patterns for each of the learned network, where testing is done on jittered single spike inputs with 1 = 10 ms. The training was done on a network consisting of m = 10 dendrites in the PDT and NDT of all NC = 10 classes. Hence, the total number of dendrites used is M = 200. As shown in Figure 4, for the rate-based learning, the training error increases while the testing error on spike versions of the inputs reduces as the training set size increases. It is clear from this result that as more data is added, it becomes diﬃcult to memorize the data. However, the generalization performance improves with more training data. A comparison between the spike-based and rate-based learning schemes is included in the Appendix, where we have shown that we can achieve similar performance for these two forms of learning. 2.1.3. Adaptive Learning Scheme for Multiclass Classiﬁcation This adaptive learning method is guided by the level of diﬃculty of each neuron’s classiﬁcation task and moreover, it is used to learn the relative size of each neuron and not the optimal neuron topology. While performing this adaptation, we keep the number of synapses per branch, k, to be a constant for FIGURE 3 \| Dendritic classiﬁer is trained adaptively by adding dendrites (step-1) and keeping the number of synapses/dendrite (k) as a constant for all neurons. The total number of synapses for the µth neuron learned in this manner (sµ adapt) is ﬁxed and the corresponding optimal topology (mµ opt, kµ opt inset plot) is theoretically determined (step-2). The length of sides of the rectangle denote m and k values and the area represents the total number of synapses, s. FIGURE 3 \| Dendritic classiﬁer is trained adaptively by adding dendrites (step-1) and keeping the number of synapses/dendrite (k) as a constant for all neurons. The total number of synapses for the µth neuron learned in this manner (sµ adapt) is ﬁxed and the corresponding optimal topology (mµ opt, kµ opt inset plot) is theoretically determined (step-2). The length of sides of the rectangle denote m and k values and the area represents the total number of synapses, s. March 2016 \| Volume 10 \| Article 113 7 Frontiers in Neuroscience \| www.frontiersin.org Structural Learning with Binary Synapses Hussain and Basu TABLE 1 \| Parameters for rate-based multiclass model. d m k nR nT Tsyn 784 10 10 25 25 100 ms TABLE 2 \| Parameters for spike-based multiclass model. τV τu τpre τpost Vthr T Tsyn 5 ms 200 ms 10 ms 200 ms 0.1 mV 200 ms 100 ms on P = 200 −20, 000 patterns using the rate-based learning scheme. Each training accuracy is obtained by averaging over 3 trials and the testing accuracy is computed by averaging across 10 presentations of 10, 000 test patterns for each of the learned network, where testing is done on jittered single spike inputs with 1 = 10 ms. The training was done on a network consisting of m = 10 dendrites in the PDT and NDT of all NC = 10 classes. Hence, the total number of dendrites used is M = 200. 3.2. Effect of Training Set Size In the ﬁrst experiment, we measured the eﬀect of training set size on the classiﬁcation performance by training the model March 2016 \| Volume 10 \| Article 113 Frontiers in Neuroscience \| www.frontiersin.org 8 Structural Learning with Binary Synapses Hussain and Basu ensemble created with a few classiﬁers (3−5), we do not know the number of dendrites required by each classiﬁer and also further, if diﬀerent levels of network complexity underlie representation of diﬀerent classes to which data belongs. Therefore, we use the adaptive learning rule to automatically learn the number of dendrites suitable for each class of a given classiﬁcation problem. These results indicate that the ensemble model can attain reasonably good performance when the member classiﬁers are trained using a moderate number of dendrites. In an ideal scenario, we can use a large number of classiﬁers to achieve signiﬁcant improvement in performance. However, it is impractical to train several classiﬁers and hence, we use only a few classiﬁers which result in most of the performance gain of the ensemble. In case of limited resources (total number of dendrites M), an intermediate level of complexity of the ensemble comprising a moderate number of member classiﬁers with a moderate number of dendrites in each classiﬁer can be used. Next, we investigate how to determine this intermediate level of complexity for a particular classiﬁcation problem. For an 3.4. Performance of the Adaptive Learning Scheme We have used the adaptive learning scheme in which P = 20, 000 patterns were used for training by initializing the network with m = 5 dendrites in each dendritic tree of a class, and then adaptively increasing m in a class-speciﬁc manner. The learning process was stopped when 150 minima were encountered and therefore, each adaptively learned classiﬁer took diﬀerent number of iterations to complete 150 minima. If dendrites are added to only 5 worst-performing classes whenever their learning slows down, referred to as scheme-1, the accuracy obtained on 10, 000 test single spike inputs is 92.1%. The number of dendrites learned by each class in one simulation run is shown by the bar plot at the top of Figure 6A. It can be seen that the digits “2,” “3,” “5,” “8,” and “9” use most of the dendrites and hence are most diﬃcult to learn while “0,” “1,” and “6” are the easier ones requiring only a small number of dendrites. The confusion matrix at the bottom of Figure 6A shows the classiﬁcation accuracy when an actual digit (column-wise) is predicted by the model as represented row- wise. We can see that the neurons for easier digits “0,” “1,” and “6” can attain good accuracy by utilizing small number of dendrites whereas the diﬃcult digits like “8” and “9” exhibit lower accuracy, where “9” is mostly misclassiﬁed as “4” and “7” having similar features. FIGURE 4 \| Training and testing error rates for rate-based learning as a function of the training set size. Test performance is measured on 10, 000 jittered single spike input patterns with 1 = 10 ms. Each data point is obtained by averaging over 3 training simulation trials and testing on 10 randomly drawn test sets for each learned network; m = 10 and k = 10. A drawback of scheme-1 is that we are not allocating the resources appropriately in this case since adding dendrites to only “diﬃcult to learn” classes might not be helpful beyond a point and adding dendrites to the easier classes also can allow the learning to converge faster and attain better performance. This idea was supported by our results obtained by adding dendrites to all the FIGURE 4 \| Training and testing error rates for rate-based learning as a function of the training set size. 3.4. Performance of the Adaptive Learning Scheme Training set consists of 20, 000 binary digit samples and testing done on 10, 000 jittered single spikes (1 = 10 ms). Model was initialized with m = 5 dendrites for all the classes. FIGURE 6 \| (A) The number of dendrites learned using adaptive scheme-1 (top) and the resulting confusion matrix (bottom) showing higher accuracy for easier classes (0, 1, 6) and lower accuracy for difﬁcult digits (8, 9). (B) Number of dendrites learned using adaptive scheme-2 (top) demonstrating all the class neurons adding dendrites when required. Confusion matrix (bottom) showing improved accuracy for all the classes. Training set consists of 20, 000 binary digit samples and testing done on 10, 000 jittered single spikes (1 = 10 ms). Model was initialized with m = 5 dendrites for all the classes. FIGURE 6 \| (A) The number of dendrites learned using adaptive scheme-1 (top) and the resulting confusion matrix (bottom) showing higher accuracy for easier classes (0, 1, 6) and lower accuracy for difﬁcult digits (8, 9). (B) Number of dendrites learned using adaptive scheme-2 (top) demonstrating all the class neurons adding dendrites when required. Confusion matrix (bottom) showing improved accuracy for all the classes. Training set consists of 20, 000 binary digit samples and testing done on 10, 000 jittered single spikes (1 = 10 ms). Model was initialized with m = 5 dendrites for all the classes. FIGURE 6 \| (A) The number of dendrites learned using adaptive scheme-1 (top) and the resulting confusion matrix (bottom) showing higher accuracy for easier classes (0, 1, 6) and lower accuracy for difﬁcult digits (8, 9). (B) Number of dendrites learned using adaptive scheme-2 (top) demonstrating all the class neurons adding dendrites when required. Confusion matrix (bottom) showing improved accuracy for all the classes. Training set consists of 20, 000 binary digit samples and testing done on 10, 000 jittered single spikes (1 = 10 ms). Model was initialized with m = 5 dendrites for all the classes. FIGURE 7 \| Error rate of ensemble consisting of NLD classiﬁers trained on P = 20, 000 MNIST samples using the adaptive scheme-2. The ensemble accuracy on 10, 000 test patterns is computed for binary vectors and spike inputs with different levels of noise. The accuracy obtained for single spike inputs with time window size 1 = 20 ms is 96.1% by combining 5 classiﬁers. 3.4. Performance of the Adaptive Learning Scheme For Poisson spike inputs, fhigh = 250 Hz and flow = 1 Hz. Each data point is obtained by randomly selecting a subset (N) of the total number of learned classiﬁers (5). This process is repeated 5 times and the average of the test errors of the combined classiﬁers is computed. classes, referred to as scheme-2, which yielded a classiﬁcation accuracy of 94.2%. The number of dendrites learned by all classes is shown at the top of Figure 6B, indicating that the dendrites are added to not only the diﬃcult classes but the easier digits like “0,” “1,” and “6” also utilize more resources. The confusion matrix shows that the classiﬁcation accuracy of almost all the digits increase as compared to scheme-1, thereby contributing to the overall improved performance. Frontiers in Neuroscience \| www.frontiersin.org 3.5. Test of Noise Sensitivity We have also tested the performance of an ensemble created with classiﬁers learned using the adaptive scheme-2, in the presence of noisy spike inputs. Figure 7 shows the classiﬁcation accuracy for 10, 000 test patterns consisting of single spike inputs with diﬀerent levels of jitter 1 and Poisson spike inputs with fhigh = 250 Hz and flow = 1 Hz. For reference, we also present the testing accuracy obtained when non-spiking binary inputs are used (solid blue), which increases from 94.6% to 96.1% by adding just 3 classiﬁers to the ensemble and further increases to about 96.4% by adding 2 more classiﬁers. The accuracy for single spikes with no noise (1 = 0) is about 0.2 −0.5% less than that of binary vectors, which can be attributed to the eﬀects of integrate and ﬁre process of the neurons in the model. As noisy single spikes are presented, accuracy reduces further. However, the change is only about 0.5% for 1 = 20 ms as compared to 1 = 0. Further, the performance of the ensemble on spiking inputs with and without noise become very similar when just 3 dendritic classiﬁers are combined together. Similarly, for Poisson spike inputs the ensemble yields similar classiﬁcation accuracy as in the absence of noise. Hence, the ensemble consisting of a FIGURE 7 \| Error rate of ensemble consisting of NLD classiﬁers trained on P = 20, 000 MNIST samples using the adaptive scheme-2. The FIGURE 7 \| Error rate of ensemble consisting of NLD classiﬁers trained on P = 20, 000 MNIST samples using the adaptive scheme-2. The ensemble accuracy on 10, 000 test patterns is computed for binary vectors and spike inputs with different levels of noise. The accuracy obtained for single spike inputs with time window size 1 = 20 ms is 96.1% by combining 5 classiﬁers. For Poisson spike inputs, fhigh = 250 Hz and flow = 1 Hz. Each data point is obtained by randomly selecting a subset (N) of the total number of learned classiﬁers (5). This process is repeated 5 times and the average of the test errors of the combined classiﬁers is computed. ensemble accuracy on 10, 000 test patterns is computed for binary vectors and spike inputs with different levels of noise. The accuracy obtained for single spike inputs with time window size 1 = 20 ms is 96.1% by combining 5 classiﬁers. 3.4. Performance of the Adaptive Learning Scheme Test performance is measured on 10, 000 jittered single spike input patterns with 1 = 10 ms. Each data point is obtained by averaging over 3 training simulation trials and testing on 10 randomly drawn test sets for each learned network; m = 10 and k = 10. FIGURE 5 \| (A) Ensemble error rate as a function of the number of classiﬁers combined, N, for 10, 000 test samples; m = 2 and k = 10. (B) Ensemble error rate as a function of the total number of dendrites used, M, showing that a moderate number of dendrites per dendritic tree (m = 8) gives reasonably good performance and there is no signiﬁcant improvement when m is further increased. Training done on P = 5000 patterns randomly drawn from the MNIST samples. Each data point is obtained by randomly selecting a subset (N) of the total number of learned classiﬁers for each case of m (37 for m = 2). This process is repeated 10 times and the average of the test errors of the combined classiﬁers is computed. FIGURE 5 \| (A) Ensemble error rate as a function of the number of classiﬁers combined, N, for 10, 000 test samples; m = 2 and k = 10. (B) Ensemble error rate as a function of the total number of dendrites used, M, showing that a moderate number of dendrites per dendritic tree (m = 8) gives reasonably good performance and there is no signiﬁcant improvement when m is further increased. Training done on P = 5000 patterns randomly drawn from the MNIST samples. Each data point is obtained by randomly selecting a subset (N) of the total number of learned classiﬁers for each case of m (37 for m = 2). This process is repeated 10 times and the average of the test errors of the combined classiﬁers is computed. March 2016 \| Volume 10 \| Article 113 Frontiers in Neuroscience \| www.frontiersin.org 9 Structural Learning with Binary Synapses Hussain and Basu FIGURE 6 \| (A) The number of dendrites learned using adaptive scheme-1 (top) and the resulting confusion matrix (bottom) showing higher accuracy for easier classes (0, 1, 6) and lower accuracy for difﬁcult digits (8, 9). (B) Number of dendrites learned using adaptive scheme-2 (top) demonstrating all the class neurons adding dendrites when required. Confusion matrix (bottom) showing improved accuracy for all the classes. 3.6. Analysis of Dendrite Weights: Feature Maps p We also analyzed the weights of dendrites to understand the features of inputs learned by the nonlinear dendrites. Since our learning rule encourages correlated inputs to be grouped together on the same dendrite, we expected the dendrite weight maps to capture the input correlations. For this purpose, we mapped the d = 784-dimensional binary weight vector w.j = [w1jw2j . . . wdj] for the jth dendrite to a 28 × 28 matrix consisting of binary elements (since there can be multiple connections from the same aﬀerent on a dendrite, the weight vector can have integer values > 1). For this analysis, 20, 000 patterns were trained using the adaptive scheme-2 by initializing the network with m = 5 dendrites for each class. The weight matrices are visualized as shown in Figure 8. The ﬁrst row depicts the mean weights of all the dendrites corresponding to a digit. It can be seen that the dendrites learn the features of digit images presented to the network. The dendrites belonging to a particular digit class together represent the complete digits. The feature maps look pixelated due to sparse integer weights learned by each dendrite. The remaining rows show the weights of 4 individual dendrites, m1 to m4, randomly selected out of the total learned dendrites for each digit. These maps demonstrate that each dendrite learns some features of the input digits. However, some of the dendrites like m4 of digit “3” and m3 of digit “7” can learn to represent complete digits, which suggests that some of the dendrites for these classes are redundant and hence, can be removed. A hybrid approach which involves adding new dendrites and pruning the superﬂuous ones can therefore be used to design the network. 3.5. Test of Noise Sensitivity For Poisson spike inputs, fhigh = 250 Hz and flow = 1 Hz. Each data point is obtained by randomly selecting a subset (N) of the total number of learned classiﬁers (5). This process is repeated 5 times and the average of the test errors of the combined classiﬁers is computed. few classiﬁers where each classiﬁer is individually trained using the adaptive learning scheme oﬀers the following advantages: (1) performance gain; (2) feasible to train few classiﬁers; (3) automatically learned network complexity; (4) noise sensitivity March 2016 \| Volume 10 \| Article 113 Frontiers in Neuroscience \| www.frontiersin.org 10 Structural Learning with Binary Synapses Hussain and Basu and (5) limited resources due to the use of a few classiﬁers with adaptively learned network size. learning rule. Figure 9C shows the comparison between the performance of our adaptively learned network (blue) and the corresponding theoretically optimal network (red). These classiﬁers were combined in ensembles and tested on 10, 000 non-spiking binary inputs and jittered single spike patterns. The results show that the optimal topology can achieve about 0.5 −0.9% higher accuracy than the adaptively learned conﬁguration. Moreover, the use of optimal m and k values also helps to generate the highest accuracy of our model, 96.6%, obtained on the binary test inputs. Further advantage of using a network with optimal topology can be understood by comparing these results with the accuracies depicted in Figure 7. We can see that the ensemble of 3 classiﬁers with optimal network topology can attain higher accuracy than the ensemble of 5 adaptively learned classiﬁers. Hence, theoretical capacity predictions of the optimal network conﬁguration can be used to boost the classiﬁcation performance of our structural learning rule while using same synaptic resources as the adaptive case. 3.8. Classiﬁcation of Event-based MNIST-DVS Dataset We also evaluate the performance of our adaptive learning algorithm on the actual event-based MNIST dataset consisting of dynamic vision sensor (DVS) recordings of diﬀerent handwritten digits (Serrano-Gotarredona and Linares-Barranco, 2014). This dataset was generated by using 10, 000 of 28 × 28 pixel digit images from the original MNIST dataset which were enlarged to three diﬀerent scales using smoothing interpolation algorithms. These upscaled digit images were displayed on an LCD monitor with slow motion and a 128 × 128 pixel AER DVS (Serrano- Gotarredona and Linares-Barranco, 2013) was used to record these moving digits for a total time duration of 2 s. We used 10, 000 recordings of moving digits upscaled to scale-4 for our analysis, which were also used to evaluate the performance of the event-driven categorization system proposed in Zhao et al. (2015). The training was performed on randomly selected 90% of the total 10, 000 recordings while the remaining 10% recordings were used for testing. For training, the event streams for 128×128 DVS recordings were converted to 128 × 128 pixel images by calculating the total number of events occurring at each pixel location for two diﬀerent time durations, 100 ms and full length of 2 s. The 128 × 128 images were then cropped to digit patches by selecting the location of 28×28 squares with maximum events occurring. Frontiers in Neuroscience \| www.frontiersin.org 3.7. Boosting the Performance using Optimal Neuron Topology consisting of spiking Restricted Boltzmann Machines (RBM) map the weights learned using oﬄine rate-based scheme onto spiking neural network in O’Connor et al. (2013) and use a spike- based event-driven learning rule based on STDP in Neftci et al. (2014). In Brader et al. (2007), a stochastic spike-driven synaptic plasticity rule was used to train a network of binary synapses and classiﬁcation accuracy determined by voting over a pool of neurons. Table 4 compares the training, test sizes, number of neurons or dendrites, synapses used and the accuracy attained by 3.7. Boosting the Performance using Optimal Neuron Topology p p gy Next, we determine the theoretically optimal conﬁguration of the network which can be used to boost the performance of our structural learning scheme, as discussed in Section 2.1.3. For this analysis, we used one instance of the ﬁnal number of dendrites learned for each class using the adaptive scheme-2 (Figure 6B). We then computed the theoretical capacity for each neuron of all classes by ﬁxing the total number of synapses learned by the µth neuron, sµ adapt, and varying the m and k values such that sµ adapt = sµ opt = m × k (step-2 in Figure 3). Figure 9A shows the theoretical capacity of class “5” neuron plotted as a function of m, where m5 adapt is the number of dendrites learned using the proposed adaptive method while m5 opt is the number of dendrites corresponding to the maximum capacity for a neuron with the same s5 opt = s5 adapt as that of a neuron trained adaptively. The number of dendrites, m7 adapt learned by our method and the corresponding optimal value, m7 opt for class “7” neuron are shown in Figure 9B. The rate-based adaptive scheme was used for training followed by testing on event streams occurring at the 28 × 28 patch extracted from the original 128 × 128 DVS recordings. The training procedure was repeated 3 times and the average testing accuracy over 3 trials was determined as shown in Table 3, which also compares with the performance of the event driven system in Zhao et al. (2015). The results show that our adaptive dendritic algorithm can achieve comparable testing accuracy (88.1% for 2 s) with the other reported performance on MNIST-DVS dataset, with higher accuracy attained when longer recordings are used. Moreover, our method yields lower training accuracy than Zhao We then trained a network with neurons consisting of mµ opt dendrites on 20, 000 MNIST samples using our structural March 2016 \| Volume 10 \| Article 113 Frontiers in Neuroscience \| www.frontiersin.org 11 Structural Learning with Binary Synapses Hussain and Basu et al. (2015) while the testing accuracy is similar, suggesting that our method is more robust to overﬁtting. et al. (2015) while the testing accuracy is similar, suggesting that our method is more robust to overﬁtting. 3.9. Comparison with Other Spike-based Classiﬁcation Models Finally, we compare the performance of our model with other spike classiﬁcation models consisting of a network of spiking neurons. The spike classiﬁers considered here use the two main approaches of rate-based and spike-based learning. The models FIGURE 8 \| Weight maps of all dendrites of neurons belonging to a particular digit class (top row). Maps in the remaining rows show individual dendrite weights with completely or partially learned features of input digits. FIGURE 8 \| Weight maps of all dendrites of neurons belonging to a particular digit class (top row). Maps in the remaining rows show individual dendrite weights with completely or partially learned features of input digits. FIGURE 8 \| Weight maps of all dendrites of neurons belonging to a particular digit class (top row). Maps in the remaining rows show individual dendrite weights with completely or partially learned features of input digits. FIGURE 9 \| (A) Theoretical capacity of neuron corresponding to digit “5” as a function of number of dendrites (m). The number of dendrites learned adaptively is m5 adapt and the optimal number of dendrites corresponding to maximum capacity is m5 opt. (B) Adaptively learned and optimal m values for digit “7.” (C) Performance comparison of adaptively learned network (blue) with theoretically determined optimal network (red). Training was done on 20, 000 binary digit samples and test performance was measured on 10, 000 binary inputs (solid) and jittered single spike input patterns with 1 = 10 ms (dashed). FIGURE 8 \| Weight maps of all dendrites of neurons belonging to a particular digit class (top row). Maps in the remaining rows show individual dendrite weights with completely or partially learned features of input digits. FIGURE 9 \| (A) Theoretical capacity of neuron corresponding to digit “5” as a function of number of dendrites (m). The number of dendrites learned adaptively is m5 adapt and the optimal number of dendrites corresponding to maximum capacity is m5 opt. (B) Adaptively learned and optimal m values for digit “7.” (C) Performance comparison of adaptively learned network (blue) with theoretically determined optimal network (red). Training was done on 20, 000 binary digit samples and test performance was measured on 10, 000 binary inputs (solid) and jittered single spike input patterns with 1 = 10 ms (dashed). FIGURE 9 \| (A) Theoretical capacity of neuron corresponding to digit “5” as a function of number of dendrites (m). 4.1. Role of Dendritic Nonlinearity in Neuronal Processing For our model, each dendrite can be considered as a processing subunit and is therefore also shown here. For the other networks, all synaptic currents sum up linearly implying the use of only one dendritic branch per neuron. The number of training examples used by Brader et al. (2007) and Neftci et al. (2014) and our method is 20, 000 while the training set in O’Connor et al. (2013) consisted of 120, 000 samples generated by introducing small random translations, rotations and scalings in the original MNIST training examples. The number of test patterns used by all the models is 10, 000. The results suggest that the NLD model can achieve accuracy comparable with other algorithms by utilizing signiﬁcantly less, which is about 2 −7% of the total number of synapses used by the other methods. Moreover, the use of low resolution integer weights in our work in contrast with the high resolution weights used by spiking RBM models (Neftci et al., 2014; O’Connor et al., 2013) renders our structural learning rule for NLDs more feasible for implementing spike classiﬁcation in hardware. Further, the ensemble of NLD classiﬁers yielded 96.1% accuracy by using rate- based learning and testing on Poisson spike inputs. Moreover, the use of optimal network topology, which was determined using the theoretical capacity calculations, enabled us to attain even higher accuracy by reducing the synaptic resources by about 1.7 times. These results are at par with the best performance achieved by Brader et al. (2007). It is important to note that both models in Brader et al. (2007) and our work use binary synapses; however, we obtain similar performance as in their work by training an ensemble of only a few classiﬁers resulting in still less number of synaptic resources, about 20% of that used in Brader et al. (2007). We also expect that our learning rule will be more amenable for hardware implementation since the dendritic polynomial nonlinearity is much simpler than implementation of a full neuron as in the population of output neurons in Brader et al. (2007). Therefore, the use of much less number of synaptic resources with binary weights and a more simpler learning rule render our NLD model more hardware-friendly. 3.9. Comparison with Other Spike-based Classiﬁcation Models The number of dendrites learned adaptively is m5 adapt and the optimal number of dendrites corresponding to maximum capacity is m5 opt. (B) Adaptively learned and optimal m values for digit “7.” (C) Performance comparison of adaptively learned network (blue) with theoretically determined optimal network (red). Training was done on 20, 000 binary digit samples and test performance was measured on 10, 000 binary inputs (solid) and jittered single spike input patterns with 1 = 10 ms (dashed). March 2016 \| Volume 10 \| Article 113 Frontiers in Neuroscience \| www.frontiersin.org 12 Structural Learning with Binary Synapses Hussain and Basu TABLE 3 \| Performance on MNIST-DVS dataset. Time length of recording used Training accuracy (%) Testing accuracy (%) 100 ms (Zhao et al., 2015) 98.9 76.9 2 s (Zhao et al., 2015) 99.1 88.2 100 ms (this work) 93.7 80.2 2 s (this work) 97.3 88.1 4.1. Role of Dendritic Nonlinearity in Neuronal Processing 4.2. Structural Plasticity as a Learning Mechanism The phenomenon of structural plasticity involving formation and elimination of synapses thereby leading to alterations to the cortical wiring diagram (Butz et al., 2009; Chklovskii et al., 2004) provides for alternative form of long term information storage in addition to the traditional synaptic weight plasticity. The information storage capacity associated with structural plasticity lies in the ability to change wiring diagram in a sparsely connected network, which provides a large number of functionally distinct circuits available to encode information (Chklovskii et al., 2004) and hence has important implications for the computational properties of the network. The computational modeling study by Poirazi and Mel (2001) demonstrated the use of structural plasticity to modify binary synaptic connections on dendritic branches. Similar to our model, a poorly performing active synapse is eliminated and replaced by the best performing synapse in a set of silent synapses. However, our learning rule is simple and easier to implement in hardware systems as compared with the learning rule used by Poirazi and Mel (2001). 4.1. Role of Dendritic Nonlinearity in Neuronal Processing TABLE 3 \| Performance on MNIST-DVS dataset. Several experimental evidences support the nonlinear processing in dendrites including active backpropagation of axonal spikes into the dendritic tree and dendritic spikes (Hausser et al., 2000; Schiller et al., 2000). However, there are not many evidences regarding the role of these nonlinear mechanisms in synaptic integration in pyramidal neurons. Experimental and compartmental modeling studies of pyramidal neurons have indicated that nearby synaptic inputs on the same dendrite sum sigmoidally while inputs on diﬀerent dendrites sum up linearly (Poirazi et al., 2003b; Polsky et al., 2004). These ﬁndings support the notion of a two-layer model of neurons, thereby having implications for the synaptic plasticity and the computational capacity of cortical tissue. Mel and his group presented several computational studies to elucidate the role of dendrites in neuronal processing. (Mel, 1991; Poirazi et al., 2003b). In more recent studies, an abstract two-layer model using sigmoidal dendritic nonlinearity was shown to predict the ﬁring rate of a detailed compartmental model of a pyramidal neuron (Poirazi et al., 2003a) and much larger storage capacities were computed for dendritic neurons with degree 10 polynomial nonlinearity in Poirazi and Mel (2001). In contrast to these studies, we use a more hardware-friendly quadratic nonlinearity which is easier to implement than a sigmoid or a high order polynomial. We also modify the learning rule to adapt the structure of the dendritic tree of diﬀerent neurons in the network according to diﬃculty of the classiﬁcation task. these classiﬁcation algorithms on MNIST dataset. The number of neurons mentioned is the total number of neurons in the network excluding those in the input layer. For our model, each dendrite can be considered as a processing subunit and is therefore also shown here. For the other networks, all synaptic currents sum up linearly implying the use of only one dendritic branch per neuron. The number of training examples used by Brader et al. (2007) and Neftci et al. (2014) and our method is 20, 000 while the training set in O’Connor et al. (2013) consisted of 120, 000 samples generated by introducing small random translations, rotations and scalings in the original MNIST training examples. The number of test patterns used by all the models is 10, 000. these classiﬁcation algorithms on MNIST dataset. The number of neurons mentioned is the total number of neurons in the network excluding those in the input layer. Frontiers in Neuroscience \| www.frontiersin.org 4.3. Binary Synapses: Computational Challenges Here, we discuss the neurobiological relevance of our work and its potential for future hardware implementation. We also compare our method with other studies based on these neurobiological mechanisms. Finally, we discuss the future directions of our work. There is accumulating experimental evidence that biological synapses exist in only a small number of states which can be restricted to even two states (O’Connor et al., 2005; Petersen et al., 1998). The use of synapses with only one or two bits of long-term March 2016 \| Volume 10 \| Article 113 Frontiers in Neuroscience \| www.frontiersin.org 13 Structural Learning with Binary Synapses Hussain and Basu TABLE 4 \| Comparison with spike classiﬁers on MNIST data. Model #Train #Test #Neurons #Dendrites #Synapses Accuracy % O’Connor et al., 2013 120, 000 10, 000 1010 1010 647, 000 94.09 Neftci et al., 2014 20, 000 10, 000 540 540 412, 000 91.9 Brader et al., 2007 20, 000 10, 000 150 150 117, 600 96.5 Adaptive NLD 20, 000 10, 000 20 440 6720 94.2 Ensemble NLD 20, 000 10, 000 100 2312 35, 285 96.1 Optimal NLD 20, 000 10, 000 100 3960 20, 163 96.4 TABLE 4 \| Comparison with spike classiﬁers on MNIST data. In most current event-based neuromorphic systems, this connection matrix is stored in a separate memory (Liu, 2014) either on or oﬀchip. This implies that since our hardware architecture enforces sparsity, we require less memory and memory reads to store and access connection information respectively. Before expanding on this point, it is important to note that we are not considering advantages of hardware implementations of on-chip learning to ﬁnd optimal connections (though we have presented some initial results on the same in Roy et al., 2014b). We are only comparing the advantages of using our proposed architecture to implement the ﬁnal network and using structural plasticity to reduce the memory requirement of this implementation. In this context, it should be noted that normal weight learning methods do not necessarily produce sparse weights and simple quantization of small weights to zero values increase errors. This was shown to be true for an ensemble of perceptrons trained by the p-delta algorithm in Roy et al. (2014a). More recently, there have been eﬀorts to improve rounding algorithms to reduce weight resolution for eﬃcient implementation of deep networks (Muller and Indiveri, 2015). 4.3. Binary Synapses: Computational Challenges Even with these methods, a two layer fully connected neural network with 500 hidden nodes needs at least 4 bits per synaptic weight to achieve comparable performance (∼96%) as our network on the MNIST dataset. This results in approximately 397, 000 4-bit weights as opposed to ∼21, 000 1-bit weights in our case. information has severe implications for the storage capacity of networks working as classiﬁers or associative memories with capacity for binary synapses reducing by more than half as compared to the capacity using continuous-valued synapses (Senn and Fusi, 2005). Some studies have presented learning algorithms as biological solutions to deal with the reduced storage capacity of networks with binary synapses. A stochastic spike- driven synaptic plasticity rule was used to train a network of binary synapses, where a pool of output neurons was used to calculate the classiﬁcation accuracy by a voting mechanism (Brader et al., 2007). This results in a large number of synapses being used. In comparison to this study, our model employs a sparsely connected network of binary synapses which learns by using a correlation-based structural plasticity rule. The use of dendritic nonlinearity yields higher computational power thereby alleviating the problem of reduced capacity of binary weights. Also, the adaptive learning of number of dendrites according to problem complexity reduces the number of synapses compared to a brute force approach. Hence, our model can achieve higher accuracy by utilizing a small number of binary synapses. Frontiers in Neuroscience \| www.frontiersin.org 4.4. Binary Synapses and Structural Plasticity: Considerations for Hardware Implementation Over the past decade, several low-power neuromorphic systems have been built to perform classiﬁcation of spike patterns. A common feature in several of these systems is the usage of binary synapses (Arthur and Boahen, 2007; Indiveri et al., 2006; Mitra et al., 2009). One reason for this is the ease with which two states can be stored in current CMOS technology using a latch. This also makes the system more robust to parametric variations due to mismatch in device - it is unlikely that high resolution weights can be obtained from a massive array of analog synapses due to a combination of systematic and random mismatch (Linares- Barranco et al., 2003). Even a recently introduced multi-core asynchronous digital chip (Merolla et al., 2014) uses a limited number of weight values per axon per core. Our algorithm is consistent with this philosophy of low-resolution weights since we limit the number of synaptic connections per dendrite and each connection is a binary value. Eﬀectively each input aﬀerent (or axon) connects with a small integer weight to a dendrite. To generalize this result, let us consider a two layer network for the conventional case with d inputs, H hidden layer neurons and C output neurons for “C” classes. The comparable network in our proposed case has H dendrites and C output neurons. “2m” out of the H dendrites connect to each of the C output neurons (H = 2m × C) using unit weights and hence can be implicitly implemented by accumulators. Considering each weight of the conventional network having resolution of “b” bits, the total number of bits required by the conventional network (NOBconv) is given by: NOBconv = b×H×d+b×H×C ≈b×H×d for C << d (22) For the proposed case, the connection matrix is of size d × H though only k × H entries are non-zero where k << H. To implement this sparse connectivity eﬃciently in an address event framework, we propose to use a two tier addressing scheme as shown in Figure 10. Here, the incoming address will be used to index into a pointer array of “d” entries with ⌈log2(H×k)⌉bits per For the proposed case, the connection matrix is of size d × H though only k × H entries are non-zero where k << H. 4.4. Binary Synapses and Structural Plasticity: Considerations for Hardware Implementation FIGURE 10 \| To implement the sparse connectivity matrix, a two level addressing scheme is proposed where the ﬁrst memory (indexed by the incoming address of the event) holds pointers to valid connection addresses stored in the second memory. (23) NOBprop = d × ⌈log2(H × k)⌉+ H × k × ⌈log2(H)⌉ (23) The memory requirements for proposed and conventional methods are compared in Figure 11 by setting b = 4, H = 104 and varying d over a wide range for k = 16, 32, and 64. It can be seen that the proposed method requires much less memory than the conventional case for large values of d when the sparsity is higher while the overhead of having a pointer array is more for small values of d. The crossover typically happens for d < 200 for values of k as large as 64. Since for most practical cases d is much larger, we expect our method to be widely applicable. The memory requirements for proposed and conventional methods are compared in Figure 11 by setting b = 4, H = 104 and varying d over a wide range for k = 16, 32, and 64. It can be seen that the proposed method requires much less memory than the conventional case for large values of d when the sparsity is higher while the overhead of having a pointer array is more for small values of d. The crossover typically happens for d < 200 for values of k as large as 64. Since for most practical cases d is much larger, we expect our method to be widely applicable. FIGURE 11 \| Memory requirement of proposed sparse connectivity scheme is compared with that of a normal fully connected weight memory with 4 bits per weight. The proposed scheme is far more memory efﬁcient for high dimensional inputs (d > 200). To underline the importance of this memory reduction, we consider a digital implementation of this system following principles similar to the ones in Merolla et al. (2014) and Seo et al. (2011). In particular, we assume that the dendritic nonlinearity/hidden neuron can be a shared physical circuit that can be time multiplexed across all required instances and we assume synaptic weight resolution is 4 bits following Seo et al. (2011). Here, for simplicity we ignore the overhead needed if the network is spread across multiple cores. 4.4. Binary Synapses and Structural Plasticity: Considerations for Hardware Implementation Using the numbers quoted in Merolla et al. (2014), the area requirement of a neuron circuit is 2900 µm2. Compared to that, the area required to implement a conventional crossbar of 4 bit weights for the MNIST case of d = 784 and say H = 1000 is 470, 400 µm2 where we estimate 0.15 µm2 area per bit from Merolla et al. (2014). This is clearly the dominant factor in chip area. Compared to this, our scheme with even k = 64, H = 1000 and d = 784 requires approximately 98, 000 µm2 area, a reduction by > 4X. FIGURE 11 \| Memory requirement of proposed sparse connectivity scheme is compared with that of a normal fully connected weight memory with 4 bits per weight. The proposed scheme is far more memory efﬁcient for high dimensional inputs (d > 200). additional source of spatial information to enhance our dendritic structural learning rule which will involve ﬁnding the optimal location on the optimal dendritic branch for a synaptic connection. The storage capacity of the network can further be increased by including distributed nonlinearity along a dendrite such that the nonlinear output of each compartment serves as input to the next compartment on the dendrite. This scheme is also more bio-realistic from the perspective of real neurons consisting of extended dendritic trees with complex branching patterns. We will also enhance our adaptive learning rule to prune the redundant or least “salient” synapses. This pruning method combined with the progressive addition of dendrites will yield an optimally sized network that will ﬁt the data. The network will learn both the number of required dendrites as well as the number of synapses on each dendrite. This approach to obtain the smallest network can also improve the generalization performance. 4.4. Binary Synapses and Structural Plasticity: Considerations for Hardware Implementation To implement this sparse connectivity eﬃciently in an address event framework, we propose to use a two tier addressing scheme as shown in Figure 10. Here, the incoming address will be used to index into a pointer array of “d” entries with ⌈log2(H×k)⌉bits per Another advantage of our architecture is that the learning happens by modifying connectivity patterns of the network. March 2016 \| Volume 10 \| Article 113 14 Structural Learning with Binary Synapses Hussain and Basu s n s n s s e s y ) ) al = 4. s n y r al y n, g d c at e l. if s n o e FIGURE 10 \| To implement the sparse connectivity matrix, a two level addressing scheme is proposed where the ﬁrst memory (indexed by the incoming address of the event) holds pointers to valid connection addresses stored in the second memory. FIGURE 11 \| Memory requirement of proposed sparse connectivity scheme is compared with that of a normal fully connected weight memory with 4 bits per weight. The proposed scheme is far more memory efﬁcient for high dimensional inputs (d > 200). entry. An incoming spike address, say “i,” is used to index into this array and read the two consecutive values ai and ai+1. As shown in the ﬁgure, suppose ai = p and ai+1 = q. ni = ai+1 −ai is the number of synapses connected to this input. If ni > 0, then ai = p is used as a pointer to the pth location in a dendrite address array. 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Nature 431, 782–788. doi: 10.1038/nature03012 LeCun, Y. AUTHOR CONTRIBUTIONS encoding of the binary images from the MNIST dataset. In a recent work (Roy et al., 2015), we have used structural plasticity to learn binary classiﬁcation spatiotemporal patterns as used in Gutig and Sompolinsky (2006). Hence, a natural extension of our present work is to combine the use of spike timing information with structural learning to enable classiﬁcation of multiclass temporal codes. SH and AB developed the idea for the paper. SH did all the modeling simulations. SH analyzed the results and both authors discussed the results and contributed to the manuscript preparation. AB supervised this work. This work was supported by MOE, Singapore through grant ARC 8/13. SH and AB developed the idea for the paper. SH did all the modeling simulations. SH analyzed the results and both authors discussed the results and contributed to the manuscript preparation. AB supervised this work. This work was supported by MOE, Singapore through grant ARC 8/13. 4.5. Future Work The classiﬁcation performance attained by our model on the benchmark MNIST data is not state-of-the-art. The best MNIST classiﬁcation result achieved so far is 99.06% accuracy using maxout networks (Goodfellow et al., 2013). Hence, we need to bridge this gap by enhancing our model. Our present model consists of lumped dendritic nonlinearity such that each dendrite is considered to be a single compartment where all the synaptic inputs are lumped together. The storage capacity of this network can be increased by introducing multiple compartments on each dendrite. The dendritic compartments represent time delays in signal propagation along a dendrite and therefore, the information about the location of synaptic inputs on a dendrite is important. We will utilize this In this work, we have not used temporal information to classify input patterns and have focussed on rate and place March 2016 \| Volume 10 \| Article 113 Frontiers in Neuroscience \| www.frontiersin.org 15 Structural Learning with Binary Synapses Hussain and Basu REFERENCES A., Arthur, J. V., Icaza, R., Cassidy, A. S., Sawada, J., Akopyan, F., et al. (2014). A million spiking-neuron integrated circuit with a scalable communication network and interface. Science 345, 668–673. doi: 10.1126/science.1254642 Hussain, S., Liu, S.-C., and Basu, A. (2014). “Improved margin multi- class classiﬁcation using dendritic neurons with morphological learning,” in Proceedings of the International Symposium on Circuits and Systems (Melbourne). doi: 10.1109/iscas.2014.6865715 Mitra, S., Fusi, S., and Indiveri, G. (2009). Real-time classiﬁcation of complex patterns using spike-based learning in neuromorphic VLSI. IEEE Trans. Biomed. 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Syst. 26, 1963–1978. doi: 10.1109/tnnls.2014.2362542 Qi, X., Guo, X., and Harris, J. (2004). “A time-to-ﬁrst spike CMOS imager,” in Proceedings of the International Symposium on Circuits and Systems (Vancouver, BC), 824–827. Reed, R. (1993). Pruning aalgorithm-a survey. IEEE Trans. Neural Netw. 4, 740– 747. Frontiers in Neuroscience \| www.frontiersin.org A. APPENDIX The correlation values averaged over all the patterns can be written as: cPDT ij =< 1cPDT ij (t) > and cNDT ij =< 1cNDT ij (t) >, where < . > indicates average calculated over an epoch. It was shown in Hussain et al. (2015) that by assuming the presynaptic time constant τpre to be much greater than the membrane time constant τV, the value of γ can be computed as: REFERENCES doi: 10.1109/72.248452 Conﬂict of Interest Statement: 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. Conﬂict of Interest Statement: 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. Roy, S., Banerjee, A., and Basu, A. (2014a). Liquid state machine with dendritically enhanced readout for low-power, neuromorphic vlsi implementations. IEEE Trans. Biomed. Circ. Syst. 8, 681–695. doi: 10.1109/TBCAS.2014.2362969 Copyright © 2016 Hussain and Basu. 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) 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. Roy, S., Kar, S. K., and Basu, A. (2014b). “Architectural exploration for on-chip, online learning in spiking neural networks,” in IEEE International Symposium on Integrated Circuits (Singapore), 128–131. doi: 10.1109/isicir.2014.70 29541 Roy, S., San, P. P., Hussain, S., Wei, L. W., and Basu, A. (2015). Learning spike time codes through morphological learning with binary synapses. IEEE Trans. March 2016 \| Volume 10 \| Article 113 Frontiers in Neuroscience \| www.frontiersin.org 17 Structural Learning with Binary Synapses Hussain and Basu A. Spike-based Structural Learning for Multiclass Classiﬁcation The multiclass BSTDSP is derived using the example of synchronous single-spike inputs such that each aﬀerent either fails to ﬁre or ﬁres a spike at a ﬁxed time Tsyn and these spike inputs are assigned to NC classes. The single-spike input is denoted by the presynaptic spike train sij(t) at the ith synapse of the jth dendrite, and the postsynaptic spike train is denoted by r(t) given by: γ = exp(−Tsyn/τpost) exp(−Trise/τpre) (A6) (A6) r(t) = X tpost δ(t −tpost) (A1) (A1) where the presynaptic spike arrives at time Tsyn and Vm(t) crosses Vst at time Trise. The connection changes are done on the basis of cij values using the same process as discussed for rate-based learning. The use of a subthreshold voltage Vst leads to a margin δspike for classifying spike patterns. A desired margin δspike for a particular class µ can also be preset, which can then be used to calculate the class speciﬁc values of Vst and Vreset as explained in Hussain et al. (2015). The class margins suited for a particular problem can be assigned by using margins set for rate-based learning. The value of the desired margin, δspike is determined by using the spike equivalent value of δ. This is done by computing the diﬀerence in the membrane voltage of the neuron (analogous to aPDT −aNDT) when a single synapse is activated. This value 1Vm multiplied by δ gives δspike, since in the binary input case the synaptic strength is normalized to 1. where the presynaptic spike arrives at time Tsyn and Vm(t) crosses Vst at time Trise. The connection changes are done on the basis of cij values using the same process as discussed for rate-based learning. The use of a subthreshold voltage Vst leads to a margin δspike for classifying spike patterns. A desired margin δspike for a particular class µ can also be preset, which can then be used to calculate the class speciﬁc values of Vst and Vreset as explained in Hussain et al. (2015). The class margins suited for a particular problem can be assigned by using margins set for rate-based learning. The value of the desired margin, δspike is determined by using the spike equivalent value of δ. This is done by computing the diﬀerence in the membrane voltage of the neuron (analogous to aPDT −aNDT) when a single synapse is activated. Frontiers in Neuroscience \| www.frontiersin.org A. APPENDIX not use postsynaptic spike–instead, the relevant event is when the membrane voltage Vm(t) crosses a subthreshold voltage Vst from below, where 0 < Vst < Vthr. These subthreshold crossing events occurring at times tst are denoted by rst(t) = P tst δ(t−tst). The correlation value is decreased when presynaptic spike time ts ij occurs before tst. The reason for using rst(t) instead of r(t) is to enforce a margin as will be explained later in this section. Finally, the change in correlation value 1cij(t) for every pattern presentation is computed using these plasticity conditions in a branch-speciﬁc manner. Hence, the learning rule for the PDT and NDT belonging to the µth class can be written as: We have proposed a biologically realistic branch-speciﬁc spike- time dependent structural plasticity (BSTDSP) rule for binary classiﬁcation in Hussain et al. (2015), which was inspired from a recent study in which reverse spike-timing dependent plasticity (RSTDP) in concert with hyperpolarization of the postsynaptic neuron was used to modify the synaptic weights (Albers et al., 2013). Our learning rule involving modiﬁcation of connections instead of synaptic weights, is in contrast with Albers et al. (2013) and is used to ﬁnd the bio-realistic correlation values cij. However, the bio-realistic basis of adapting the number of dendrites is not clear. For PDT of class µ: 1cPDT ij (t) = IPDT D,j (t)¯rPDT(t)sPDT ij (t) − γ IPDT D,j (t)¯sPDT ij (t)rPDT st (t) (A4) For NDT of class µ: 1cNDT ij (t) = INDT D,j (t)¯rNDT(t)sNDT ij (t) − γ INDT D,j (t)¯sNDT ij (t)rNDT st (t) (A5) The correlation values needed for structural plasticity rule were obtained from an online spike-based learning rule. The biological relevance of these correlations can be found in calcium concentration in spines which is a correlation sensitive, spike- time dependent signal and has been implicated as a guide for structural plasticity (Helias et al., 2008). Here, we present an extension of BSTDSP to multiclass classiﬁcation and also demonstrate the correspondence between rate-based and spike- based learning approaches. (A4) (A5) where γ is a constant used to balance the potentiation and depression and ensure that cij = 0 when a pattern is learned. where γ is a constant used to balance the potentiation and depression and ensure that cij = 0 when a pattern is learned. A. Spike-based Structural Learning for Multiclass Classiﬁcation This value 1Vm multiplied by δ gives δspike, since in the binary input case the synaptic strength is normalized to 1. where tpost is the postsynaptic spike time. These pre- and postsynaptic spikes also drive exponentially decaying memory traces–the presynaptic trace ¯s and the postsynaptic trace ¯r given by: ¯s = exp(−t/τpre) (A2) ¯r = exp(−t/τpost) (A3) (A2) (A3) If a pattern belonging to class µ is presented, a teacher signal forcing a postsynaptic spike (at time t = 1 ms) is present for the (+) neuron and absent for (−) neuron of class µ. For the remaining (NC −1) classes, opposite conditions for the teacher signal exist, i.e., teacher signal is absent for (+) neuron and present for (−) neuron. The spike-based and the rate-based structural learning rules were compared in Hussain et al. (2015) and a relationship between the correlation terms [1cij]spike and [1cij]rate for the two forms of learning was derived as given below. The spike-based structural plasticity learning rule is similar to the rate-based learning method and also involves computing correlation values cij for each synapse belonging to the PDT and NDT corresponding to class µ. This learning scheme is analogous to the RSTDP rule such that if a postsynaptic spike arrives before a presynaptic spike, then the correlation value for that synapse is potentiated. The condition for depression does [1cij]spike = exp(−Tsyn/τpost)[1cij]rate (A7) (A7) Therefore, any diﬀerences between the performance of the two learning methods can be understood on the basis of this March 2016 \| Volume 10 \| Article 113 18 Structural Learning with Binary Synapses Hussain and Basu relationship. This suggests that as the value of τpost is increased, the correlation values for the rate-based learning, [1cij]rate well approximate the correlation values for the spike-based learning, [1cij]spike. Since the two forms of learning tend to coincide with increasing τpost, therefore, we have used the faster rate-based approach instead of the computationally prohibitive spike-based learning scheme for the enhancements proposed in the model. become more similar. Therefore, we need to repeat our spike learning simulations with higher values of τpost to achieve greater agreement between the two forms of learning; (2) Secondly, our estimation of γ in Equation (A6) is based on the assumption that τpre ≫τV. B. Comparison of Spike-based Learning vs Rate-based Learning Schemes g We have compared the performance of the spike-based and rate- based learning schemes. Since spike-time-based learning takes very long and the training time increases with the size of training set, therefore, we have trained the spike model on smaller number of training patterns, 200 −1000. For both forms of learning schemes, the networks consisted of m = 10 dendrites in the PDT and NDT of all NC = 10 classes. Hence, the total number of dendrites used by both models is M = 200. Figure A1A shows the comparison between the performance of spike-based and rate-based learning schemes. Both forms of learning exhibit the trend of decreasing test error with the training set size. However, the spike-based learning method results in about 1.5 times higher errors than that for rate-based learning. This diﬀerence in performance of the two methods can be attributed to three reasons: (1) Firstly, the relationship between these two methods (Equation A7) suggests that as the value of τpost is increased, the correlation values for the two learning methods Based on this discussion, we attempted to reduce the diﬀerence between the performance of rate-based and spike- time-based learning approaches. Therefore, we increased the value of τpost from 200 to 500, 1000 and 2000 ms and τpre from 10 to 50 ms. As shown in Figure A1B, the test error for τpost = 200 ms corresponds to our prior parameter settings for P = 200 patterns (Figure A1A, blue curve). As τpost is increased, the testing performance of spike-based learning rule improves and gradually becomes closer to the testing performance of rate- based method, shown with blue line. We expect that we can achieve further agreement between the two forms of learning by getting the margin values δspike and δ to be analogous to each other or by determining the desired δspike directly in the spike domain. Hence, we have shown that the error rate for spike-based learning becomes similar to that of rate-based learning if τpost is long enough. FIGURE A1 \| (A) Comparison of performance of rate-based and spike-time-based learning schemes. The test error rates for the spike-based learning rule are about 1.5 times higher than the rate-based test errors; (B) Comparison of test error rate for P = 200 patterns learned with rate-based (blue line) and spike-based (red plot) methods as a function of τpost. A. Spike-based Structural Learning for Multiclass Classiﬁcation Hence, smaller τpre will result in a non-ideal value of γ which will give diﬀerent results for the two learning schemes; and (3) Finally, the discrepancy in results can arise if the margin setting is not exact. As discussed above, the margin for spike- based learning was set using δspike = 1Vm × δ, where a wrong estimate of the constant 1Vm will mean that δspike and δ are not truly analogous to each other, leading to diﬀerent results. Frontiers in Neuroscience \| www.frontiersin.org B. Comparison of Spike-based Learning vs Rate-based Learning Schemes Each data point is obtained by averaging over 3 training simulation trials and testing on 10, 000 single spike inputs with jitter 1 = 10 ms; m = 10 and k = 10. FIGURE A1 \| (A) Comparison of performance of rate-based and spike-time-based learning schemes. The test error rates for the spike-based learning rule are about 1.5 times higher than the rate-based test errors; (B) Comparison of test error rate for P = 200 patterns learned with rate-based (blue line) and spike-based (red plot) methods as a function of τpost. Each data point is obtained by averaging over 3 training simulation trials and testing on 10, 000 single spike inputs with jitter 1 = 10 ms; m = 10 and k = 10. March 2016 \| Volume 10 \| Article 113 Frontiers in Neuroscience \| www.frontiersin.org Frontiers in Neuroscience \| www.frontiersin.org 19
https://openalex.org/W1984981145	https://discovery.ucl.ac.uk/1354302/1/1354302.pdf	English	null	Characterisation of a Peripheral Neuropathic Component of the Rat Monoiodoacetate Model of Osteoarthritis	PloS one	2,012	cc-by	11,512	Abstract This is an open-access article distributed under the terms of the Creative Commons unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the Wellcome Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have read the journal’s policy and have the following conflicts. Pregabalin was provided as a gift from Pfizer, Sandwich, UK not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. Competing Interests: The authors have read the journal’s policy and have the following conflicts. Pregabalin was provided as a gif for this study. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: ucklmth@ucl.ac.uk In parallel to degenerative changes within the joint, a pain phenotype rapidly develops in the hindlimb ipsilateral to the injected knee, suggesting the presence of central sensitization. This phenotype has been assessed using standard behavioural measures of evoked pain, including mechanical and thermal stimuli applied to an area of referred pain on the hindpaw, as well as calibrated pressure and torque applied to the knee [8,11,12]. Novel measures employed, intended to gauge movement evoked or ongoing pain, include tests of motility, weight-bearing, grip strength and sleep disruption [9,10,12,13,14,15,16,17]. PLoS ONE \| www.plosone.org Citation: Thakur M, Rahman W, Hobbs C, Dickenson AH, Bennett DLH (2012) Characterisation of a Peripheral Neuropathic Component of the Rat Monoiodoacetate Model of Osteoarthritis. PLoS ONE 7(3): e33730. doi:10.1371/journal.pone.0033730 March 2012 \| Volume 7 \| Issue 3 \| e33730 Abstract Joint degeneration observed in the rat monoiodoacetate (MIA) model of osteoarthritis shares many histological features with the clinical condition. The accompanying pain phenotype has seen the model widely used to investigate the pathophysiology of osteoarthritis pain, and for preclinical screening of analgesic compounds. We have investigated the pathophysiological sequellae of MIA used at low (1 mg) or high (2 mg) dose. Intra-articular 2 mg MIA induced expression of ATF-3, a sensitive marker for peripheral neuron stress/injury, in small and large diameter DRG cell profiles principally at levels L4 and 5 (levels predominated by neurones innervating the hindpaw) rather than L3. At the 7 day timepoint, ATF-3 signal was significantly smaller in 1 mg MIA treated animals than in the 2 mg treated group. 2 mg, but not 1 mg, intra-articular MIA was also associated with a significant reduction in intra-epidermal nerve fibre density in plantar hindpaw skin, and produced spinal cord dorsal and ventral horn microgliosis. The 2 mg treatment evoked mechanical pain-related hypersensitivity of the hindpaw that was significantly greater than the 1 mg treatment. MIA treatment produced weight bearing asymmetry and cold hypersensitivity which was similar at both doses. Additionally, while pregabalin significantly reduced deep dorsal horn evoked neuronal responses in animals treated with 2 mg MIA, this effect was much reduced or absent in the 1 mg or sham treated groups. These data demonstrate that intra-articular 2 mg MIA not only produces joint degeneration, but also evokes significant axonal injury to DRG cells including those innervating targets outside of the knee joint such as hindpaw skin. This significant neuropathic component needs to be taken into account when interpreting studies using this model, particularly at doses greater than 1 mg MIA. Citation: Thakur M, Rahman W, Hobbs C, Dickenson AH, Bennett DLH (2012) Characterisation of a Peripheral Neuropathic Component of the Rat Monoiodoacetate Model of Osteoarthritis. PLoS ONE 7(3): e33730. doi:10.1371/journal.pone.0033730 Editor: Mark L. Baccei, University of Cincinnatti, United States of America Received November 22, 2011; Accepted February 16, 2012; Published March 21, 2012 Copyright: 2012 Thakur 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: 2012 Thakur et al. Immunohistochemistry Animals were terminally anaesthetised and transcardially perfused with chilled 4% paraformaldehyde in 0.1% phosphate buffer. Lumbar spinal cord was dissected out with ipsilateral and contralateral L3, L4 and L5 DRG and proximal sciatic nerves attached. For nerve morphometry studies, the sciatic nerve was followed until it branched into the tibial, common peroneal and sural nerves posterior to the knee joint (the tibial nerve includes the posterior articular nerve that innervates the articular structures). 5 mm samples were taken from each branch, proximal to the knee joint. Additional 5 mm samples were taken from the common peroneal nerves 10 mm distal to the knee joint. Skin biopsies were taken from the hindpaw glabrous skin proximal to the pad below the first digit, taking the full width of the plantar surface. Peripheral Neuropathy in the MIA Model of OA effective in modulating nociceptive transmission in the presence of evoked central sensitization in human subjects [21]. effective in modulating nociceptive transmission in the presence of evoked central sensitization in human subjects [21]. Weight bearing was assessed using an incapacitance tester (Linton Instrumentation, Norfolk, UK). Rats were placed in a plexiglass enclosure so that each hindpaw rested on a separate weighing plate. After 2 minutes’ habituation, the force exerted by each hind paw was measured over a 5s testing period. The first 3 sets of 5s measurements were taken and then averaged. These values were then transformed to give the percentage of total hindlimb weight borne on the ipsilateral side. Data were analyzed using One-way ANOVA or 2-way ANOVA with Bonferroni’s Multiple Comparison test for time course and between group comparisons respectively. MIA has been shown to evoke ATF-3 expression in DRG cells, which was suggested to represent damage to joint afferents [22]. Pain related hypersensitivity in this model has therefore been attributed to inflammation and degenerative changes within the knee joint, as well as a possible localised neuropathic component involving joint afferents. Here we have characterised the expression of ATF-3 (a marker of neuronal injury), peripheral innervation and spinal microgliosis following MIA treatment of rats up to 14 days after model induction. We demonstrate significantly increased expression of ATF-3, primarily in L4 and L5 DRG, after intraarticular injection of 2 mg, but not 1 mg MIA. ATF-3 upregulation in the 2 mg MIA model is associated with reduced intraepidermal nerve fibre (IENF) innervation of ipsilateral plantar hindpaw skin, spinal microgliosis and greater hindpaw mechanical hypersensitivity, indicating a significant neuropathic component at this dose which includes afferents innervating territories outside of the knee joint. Additionally, while deep dorsal horn evoked responses are reduced by pregabalin in the 2 mg MIA model, the effects of the drug are significantly less pronounced where arthritis has been induced by 1 mg MIA. Experimenters were blinded to treatment groups where saline or different doses of MIA were used. Ethics Statement All experimental procedures were approved by the UK Home Office (project license 1205) and follow the guidelines of the International Association for the Study of Pain [26]. Tissues were postfixed in 4% paraformaldeyde for 2 hours at room temperature then transferred into 20% sucrose overnight at 4uC [27]. Tissue was embedded in OCT and stored at 280uC. Transverse sections of skin were cut at 14 mm on a cryostat onto chrome alum gelatin-covered slides. DRG and transverse spinal cord sections were cut at 10 mm on a cryostat onto SuperFrost Ultra Plus slides. Animal care and model induction Male Sprague-Dawley rats (Central Biological Services, Uni- versity College London) weighing 120–140 g at the time of induction were used. Animals were anaesthetised using isofluorane and a single injection of 25 ul sterile 0.9% saline containing 2 mg or 1 mg of monosodium iodoacetate (MIA, Sigma, UK) was administered through the left patellar tendon using a 27G needle. Sham injections used ipsilateral saline only, and were assessed histologically at day 7, except for articular histology, which used contralateral saline and was assessed at day 14. Nerves were postfixed in 3% glutaraldehyde at 4uC overnight, washed in 0.1 M PB, osmicated, dehydrated, and embedded in epoxy resin (TAAB Embedding Materials). 1 mm sections were cut on an ultramicrotome and stained with toludine blue. After overnight postfixation in 4% PFA, knee joints were dissected to remove muscle and tranferred into a decalcifying buffer comprising 7% AlCl3, 5% formic acid, and 8.5% HCl for 10 hours at 4uC, before being washed in 0.1 M phosphate buffer, pH7.2. processed and embedded in paraffin wax. 6 mm sagittal sections from each condyle were cut and mounted onto microscope slides, then dried at 60uC overnight. After dewaxing and rehydrating, sequential sections were stained using Toluidine blue pH4 to visualize proteoglycans in the articular cartilage. Introduction Osteoarthritis (OA) is one of the most prevalent sources of chronic pain, affecting around 10% of men and 20% of women aged 60+ worldwide [1]. The disabling effect of osteoarthritis is not simply due to altered joint biomechanics, such as locking and crepitation, but also evoked and spontaneous pain associated with the arthritic joint [2]. Current analgesics are relatively ineffective and are associated with various gastrointestinal, cardiac and renal adverse effects [3]. The monoiodoacetate model of OA, in which a single injection of the irreversible NADPH inhibitor, sodium monoiodoacetate (MIA), is made into the joint space, provides a model of the painful and structural components of OA in rodents. The doses most frequently used are 1, 2 or 3 mg [4,5,6], with the model usually assessed up to 14 days post-induction, with some studies extending further to 30, 56 or 68 days [5,7,8]. MIA has been shown to inhibit chondrocyte metabolism, precipitating a rapid degeneration of joint integrity with features mirroring those seen clinically [5]. These include synovial thickening, loss of cartilage, formation of osteophytes and eventual fibrillation of cartilage. The inflammatory early phase of the model also features joint swelling and immune cell infiltration of the patellar fat pad, and resolves fully by day 7 [9,10]. Electrophysiological studies have demonstrated peripheral changes in the excitability of knee joint afferents as well as central changes in the evoked responses and pharmacological manipula- tion of deep dorsal horn neurones with receptive fields in the hindpaw ipsilateral to joint degeneration [11,18,19]. These deep dorsal horn neurones are subject to increased 5-HT3R activity and increased endocannabinoidergic tone [18,19]. While pregabalin, an analgesic clinically effective in a variety of neuropathic conditions [20], has only minimal effectiveness on deep dorsal horn neuronal evoked responses in sham animals, it is able to significantly reduce responses in MIA animals [19]. A similar state-dependency is seen in the action of a related drug with a similar mechanism of action, gabapentin, which is more PLoS ONE \| www.plosone.org 1 PLoS ONE \| www.plosone.org March 2012 \| Volume 7 \| Issue 3 \| e33730 Peripheral Neuropathy in the MIA Model of OA Peripheral Neuropathy in the MIA Model of OA Quantification of the number and morphology of Iba1 immunoreactive microglia in the spinal cord was performed in four sections of spinal cord at the level of L4 per animal. Microglia were quantified within 4 defined 10,000 um2 areas in the superficial dorsal horn or ventral horn. Microglia in which process length was less than double the soma diameter were classified as presenting an effector morphology, while microglia in which process length was more than double soma diameter were defined as possessing surveyor morphology, a method used previously by [29]. Cells were sampled only if the nucleus was visible within the plane of section and if cell profiles exhibited distinctly delineated borders. Spinal cord ATF-3 staining was quantified as the total number of ATF-3+ profiles present in 4 spinal cord sections per animal. Pharmacological assessment was carried out on one ipsilateral neuron only per animal. One round of testing was performed every twenty minutes, and consisted of a train of electrical stimuli followed by graded natural stimuli as described above. Following three consecutive stable control trials (,10% variation for the C- fibre evoked response) neuronal responses were averaged to give the pre-drug control values. Pregabalin (a gift from Pfizer, Sandwich, UK) was dissolved in 0.9% saline solution at a concentration of 10 mg/kg, and administered via subcutaneous injection in the scruff of the back of the neck. Previous studies indicated that this concentration significantly reduced spinal neurone evoked responses in 2 mg MIA treated, but not sham injected, rats [19]. The effect of the drug was followed for an hour, with tests carried out at 10, 30 and 50 minutes after dosing. The value of greatest change from the baseline for each metric (electrical stimuli, natural stimuli) was then found and expressed as a percentage of the predose baseline, and plotted to allow comparison of drug effect in sham, 1 mg MIA and 2 mg MIA animals. Kruskal-Wallis tests with Dunn’s posttest were used to compare drug effect for each metric. The sham and 2 mg data is included for comparison, but has previously been published in a different form [19]. Knee joint sections were imaged using an Epson perfection V700 photo flatbed scanner at 3200 dpi. Peripheral Neuropathy in the MIA Model of OA manner with coding of increasing intensity; they exhibited windup when repeatedly stimulated; and they were situated at a depth of .500 mm from the surface of the spinal cord. Four mosaic-photomicrographs of complete DRG sections per DRG were randomly selected from each animal. Both ATF-3+ and the total BIIITub+ nucleated cell profile populations were counted and their diameters measured to determine cell size distribution. Because on sectioning DRG cells will be cut into multiple profiles we refer to DRG cell profiles and not absolute DRG cell numbers when discussing quantification of ATF-3 expression. Cell counting and analysis was performed using AxioVision LE, release 4.2. A train of 16 transcutaneous electrical stimuli (2 ms wide pulses, 0.5 Hz) applied at 3 times the threshold current for C-fibre activation of the dorsal horn cell was delivered via stimulating electrodes inserted into the peripheral receptive field in the hindpaw. A post- stimulus time histogram was constructed such that responses evoked via Ab – (0–20 ms), Ad – (20–90 ms) and C-fibres (90–350 ms) were separated and quantified on the basis of latency. Responses occurring after the C-fibre latency band were taken to be the post-discharge (repetitive firing) of the WDR cell (350–800 ms). Epidermal fibers were counted at 406magnification live on the microscope according to rules set out by Lauria et al. [28]. Only nerve fibers that could be seen to cross the basement membrane between the epidermis and dermis were counted. Four skin sections were counted per animal, by a blinded experimenter. The length of the epidermis sample in mosaic-photomicrograph was measured using ImageJ and the intraepidermal nerve fiber density (IENFD) calculated as number of fibers per millimeter. Data were analyzed using paired t-test of ipsilateral vs contralateral IENF density, or unpaired t-test of 2 mg vs 1 mg IENF ipsilateral density as a percentage of contralateral density. The centre of the peripheral receptive field was also stimulated using punctate mechanical and thermal stimuli (2, 8, 26 and 60 g von Frey filaments and a water jet applied at 35, 40, 45, and 48uC). Application of each von Frey hair was separated by a minimum interval period of 50 seconds. All natural stimuli were applied for a period of 10 seconds per stimulus. Data was captured and analysed by a CED 1401 interface coupled to a computer running Spike 2 software (Cambridge Electronic Design; PSTH and rate functions). Results In all cases, quantification was performed by a single, blinded observer. Unless stated otherwise, data were analysed using One- way ANOVA with Dunnett’s or Bonferroni’s multiple comparison test for timecourse or between-group comparisons respectively. Peripheral Neuropathy in the MIA Model of OA Cartilage proteoglycan content was scored on a scale of 0–4 where 0 = no loss of cartilage proteoglycan staining relative to a normal control, 1 = minimal loss, 2 = mild loss, 3 = moderate loss and 4 = total loss of proteoglycan staining. The extent of cartilage proteoglycan loss was assessed as 1/3, 2/3 or 3/3 of the condylar surfaces and the above score was multiplied by 1, 2 or 3, respectively to give a maximum score of 12 for total loss over the whole surface [30]. For articular histology only, ipsilateral MIA treated knees were compared to contralateral saline-injected knees. Pain behaviour and cartilage loss following intra-articular MIA treatment Arthritis-associated referred pain behaviours were assessed at the hindpaw as previously described [13]. Significant hypersensi- tivity to von Frey hair application and acetone cooling were both observed by day 3 in 2 mg and 1 mg MIA animals (Figure 1A Kruskal Wallis test, P,0.05 vs preinjection baseline, n = 12 animals/group). Significant weight bearing difference between ipsilateral and contralateral hindlimb was seen following 2 mg and 1 mg MIA injection (Fig. 1B, One-way ANOVA with Dunnett’s multiple comparison test, P,0.05, n = 12 animals/group). There was no hypersensitivity on the contralateral hindpaw. Consistent with previous studies, the timecourse of weightbearing asymmetry is biphasic, with asymmetry slightly correcting at the 7d timepoint but returning at 14d [6,10]. The weight-bearing alteration in the 2 mg group was not significantly different to that in the 1 mg group (2-way ANOVA with Bonferroni’s post test, n = 12 animals/ group, P.0.05 at all time points). Behaviour Behaviour was assessed at 3, 7 and 14 days following MIA injection. Testing was preceded by a 30-min acclimatisation period. Cooling hypersensitivity was assessed using a drop of acetone applied to the plantar surface of the hind paws, both ipsilateral and contralateral to the injected knee. A marked, delayed, flinching or shaking behaviour was a positive outcome. The test was repeated a total of five times on each side with a minimum of 5 min between each application. The resulting score was doubled to give a score out of 10, allowing plotting on the same axes as mechanical hypersensitivity. Mechanical hypersen- sitivity was assessed by applying 1, 6 and 8 g von Frey filaments, (Touch-test, North Coast Medical Inc., San Jose CA, USA) 5 times to the plantar surface proximal to the digits, and 5 times to the toes of the ipsilateral and contralateral hind paws. Withdrawal responses and whole paw lifts elicited by von Frey hairs were scored as positive, with a Mean Difference Score of ipsilateral – contralateral withdrawals used for graphing and statistical analysis, which used Kruskall-Wallis test with Dunn’s or Bonferroni’s multiple comparison test for timecourse and between group comparisons respectively. DRG cells were visualized by immunostaining with antibodies raised against bIIITubulin (Promega, mouse monoclonal, 1:800) and ATF-3 (Santa Cruz, rabbit polyclonal, 1:400). Intra-epidermal nerve fibres (IENF) in skin biopsies were visualized with the pan- neuronal marker protein gene product (PGP), polyclonal rabbit PGP9.5 (Ultraclone, 1:800). Spinal cord microglia were visualized using rabbit anti-Iba1 (WAKO, 1:1000). Secondary antibodies used were anti-rabbit Cy3 (Stratech; 711-166-152; 1:500; 2.5 hr) and AlexaFluor 488 anti-mouse (Invitrogen, 1:1000, 2.5 hr). Overnight incubation of primary antibodies was preceded by incubation with normal donkey serum (Millipore Bioscience Research Reagents; S30; 1:10; 30 min). All reagents were diluted in PBS/0.2% Triton X-100/0.1% sodium azide. Immunofluorescence was visualized under a Zeiss Imager.Z1 microscope. PLoS ONE \| www.plosone.org March 2012 \| Volume 7 \| Issue 3 \| e33730 March 2012 \| Volume 7 \| Issue 3 \| e33730 2 Peripheral Neuropathy in the MIA Model of OA PLoS ONE \| www.plosone.org Electrophysiology B – incapacitance testing of 2 mg or 1 mg MIA treated animals at 3, 7 and 14d after treatment (2-way ANOVA with Bonferroni’s post test, n = 12 animals/group. P.0.05 at all time points). C – 2 mg MIA treated rat knee sagittal sections from day 14, stained with toluidine blue to visualise cartilage proteoglycan content. Fem = femoral condyl, Tib = tibial condyl. Ant = anterior aspect of knee, Post = posterior aspect. D – 1 mg MIA rat knee sections from day 14. E – saline injected contralateral control knee from day 14. doi:10.1371/journal.pone.0033730.g001 When comparing the 1 mg and 2 mg MIA groups there was significantly less mechanical hypersensitivity in the 1 mg group, with significantly fewer withdrawals to the 8 g von Frey hair at all time points compared to the 2 mg group (Fig. 1A, Kruskal Wallis test with Bonferroni’s posttest, P,0.05). No significant difference between 1 mg and 2 mg groups was apparent in the response to acetone cooling or weight bearing at any timepoint. population of cells but rather present in small, medium and large cell profiles. The total mean ATF-3 signal peaked at 20.83% of nucleated profiles quantified in L4 at day 7 (Fig. 3D). Total mean ATF-3 signal was below 5% in L3 at all time points. No ATF-3 positive nuclear profiles were present in L2 and L6 DRG (data not shown). Sham injection of saline did not induce significant ATF-3 expression at any of the levels assessed at day 7, though ATF-3 expression in naı¨ve DRG was observed very infrequently (Figure 3C, D, in ,1% of profiles counted). ATF-3 expression was significantly greater at 7 and 14 days after MIA injection compared to 7d sham in L4 (Figure 3D, One-way ANOVA with Dunnett’s multiple comparison test, n = 967d sham, 1267d MIA, 7614d MIA), and at 7 days after injection in L5 (n=1267d, P,0.01). Despite the difference in evoked behaviour, the extent of articular cartilage proteogylcan loss at 14d was not significantly different in 2 mg (Fig. 1C) vs 1 mg (Fig. 1D) groups (Table S1), consistent with existing data at this time point [32]. Note that, after the 7d MIA stage, there is no significant inflammation in the joint structures [9]. Electrophysiology Two weeks after MIA injection (post-operative days 15–19), in vivo electrophysiological studies were performed as previously described [31]. Briefly, animals were anesthetized and maintained for the duration of the experiment with isofluroane (1.5–1.7%) delivered in a gaseous mix of N2O (66%) and O2 (33%). A laminectomy was performed to expose the L4–5 segments of the spinal cord. Extracellular recordings were made from ipsilateral deep dorsal horn neurones (laminae V–VI) using parylene coated tungsten electrodes (A–M Systems, USA). The neurones included in this study met the following criteria: they had a receptive field on the plantar hindpaw; they all responded with at least 50 spikes to both light touch (8 g von frey) and noxious stimuli (60 g von Frey and 48uC heat); they responded to natural stimuli in a graded PLoS ONE \| www.plosone.org March 2012 \| Volume 7 \| Issue 3 \| e33730 3 Peripheral Neuropathy in the MIA Model of OA Figure 1. Behavioural and articular histological assessment of MIA animals. A – withdrawals in response to 1 g, 6 g or 8 g von frey hair or acetone applied to the plantar hindpaw 3, 7 or 14 days after intra-articular saline, 2 mg or 1 mg MIA injection (Kruskal Wallis test of: timecourse, P,0.05 +, P,0.01 ++, P,0.001 +++ vs preinjection baseline; inter-group comparison P,0.05 , P,0.01 * n = 12 animals/group). B – incapacitance testing of 2 mg or 1 mg MIA treated animals at 3, 7 and 14d after treatment (2-way ANOVA with Bonferroni’s post test, n = 12 animals/group. P.0.05 at all time points). C – 2 mg MIA treated rat knee sagittal sections from day 14, stained with toluidine blue to visualise cartilage proteoglycan content. Fem = femoral condyl, Tib = tibial condyl. Ant = anterior aspect of knee, Post = posterior aspect. D – 1 mg MIA rat knee sections from day 14. E – saline injected contralateral control knee from day 14. doi:10.1371/journal.pone.0033730.g001 Figure 1. Behavioural and articular histological assessment of MIA animals. A – withdrawals in response to 1 g, 6 g or 8 g von frey hair or acetone applied to the plantar hindpaw 3, 7 or 14 days after intra-articular saline, 2 mg or 1 mg MIA injection (Kruskal Wallis test of: timecourse, P,0.05 +, P,0.01 ++, P,0.001 +++ vs preinjection baseline; inter-group comparison P,0.05 , P,0.01 * n = 12 animals/group). Electrophysiology Because the degree of cartilage loss could not explain the difference in mechanical hypersensitivity when comparing the 2 and 1 mg dose of MIA at day 14, we went on to assess the extent of peripheral neural injury. As expression in the 2 mg group peaked at the 7d timepoint, this timepoint was chosen to assess ATF-3 expression in the 1 mg MIA group. ATF-3 expression in 1 mg MIA treated rats (Figure 3B) was not present at a level significantly greater than that seen in saline injected sham animals at the 7d timepoint, although a strong trend was noted. There were significantly fewer ATF-3+ profiles in 1 mg (7.6% of L4 DRG) than in 2 mg (20.83% of L4 DRG) MIA treated animals at the 7 day timepoint (Figure 3E, 1 way ANOVA with Bonferroni’s multiple comparison test, n = 967d sham, 1262 mg MIA, 1261 mg MIA, 2 mg vs 1 mg P,0.001 at L4, 2 mg vs 1 mg P,0.01 at L5, 7d sham vs 1 mg P.0.05 at L4 or L5). Increased ATF-3 expression within DRG cells and motoneurones following MIA application The expression of the transcription factor ATF-3 is a sensitive marker of neuronal insult or injury. It is expressed in DRG cells and spinal cord ventral horn motoneurons within 3d days following axotomy or crush, remaining positive for at least 28 days after nerve injury [33,34]. Immunohistochemistry for ATF-3 and BIIITub positive cell profiles in the DRG enabled approximation of the percentage of total DRG cell nuclear profiles immunopositive for ATF-3 at the level of L3, L4 and L5 3, 7 and 14 days after MIA injection (Figure 2, illustrated and quantified as % of DRG in Figure 3A). Analysis of cell size distribution showed ATF-3 expression was not restricted to one Immunohistochemistry also indicated the presence of ATF-3 positive nuclei in ventral horn motor neurones of 2 mg MIA treated animals (Figure S1). The absolute number of these ATF-3 positive nuclei was small (mean of 1.2 ATF-3+ cells per section of PLoS ONE \| www.plosone.org March 2012 \| Volume 7 \| Issue 3 \| e33730 PLoS ONE \| www.plosone.org 4 Peripheral Neuropathy in the MIA Model of OA Figure 2. Cell size distribution for BIIITubulin (green bars, left y axis) and ATF-3 (red bars, right y axis) expressing profiles in DRG L3, L4 and L5 in sham or 2 mg MIA animals 3, 7 and 14 days after injection. n = 9 sham, 7 at day 3, 12 at day 7, 7 at day 14. doi:10.1371/journal.pone.0033730.g002 Figure 2. Cell size distribution for BIIITubulin (green bars, left y axis) and ATF-3 (red bars, right y axis) expressing profiles in DRG L3, L4 and L5 in sham or 2 mg MIA animals 3, 7 and 14 days after injection. n = 9 sham, 7 at day 3, 12 at day 7, 7 at day 14. doi:10.1371/journal.pone.0033730.g002 L4 spinal cord at 7d after MIA injection), but was significant at day 7 (one-way ANOVA with Dunnett’s multiple comparison test, n = 8 animals per timepoint, p,0.05). ATF-3 positive ventral horn nuclear profiles were not present in the 1 mg MIA or 7d sham treated groups. peripheral neuropathies. PGP9.5 immunoreactive IENFs were quantified in skin sections from the ipsilateral and contralateral plantar hindpaw in a blinded fashion (Figure 4A, B). Increased ATF-3 expression within DRG cells and motoneurones following MIA application 2 mg MIA induction was associated with a significant decrease in ipsilateral IENFD of 26% at day 7 and 37% at day 14 (Figure 4C, paired t- test of ipsi vs contralateral density, n = 867d, 4614d, P,0.05). The cell bodies of afferent fibres innervating the joint reside in primarily L3 and L4 DRG [35], and comprise below 5% of the total cell bodies in the DRG at these levels [36]. As the magnitude of ATF-3 expression observed in L5 DRG could not arise purely from joint afferents, we studied nerve morphometry as well as primary afferent terminations within hindpaw plantar epidermis. In 1 mg MIA treated rats there was no evidence of ipsilateral reduction in IENFD (P.0.05 vs contra side, paired t test). The ipsilateral IENFD was significantly higher than the ipsilateral 2 mg IENF density at the same time point (unpaired t test, P,0.01, n = 867d 2 mg, 467d 1 mg). PLoS ONE \| www.plosone.org Intra-articular MIA is associated with a reduction in ipsilateral hindpaw intra-epidermal nerve fibre density p g In a wide range of models, nerve injury is associated with increased numbers of microglia within the spinal cord [38,39]. Treatment with intra-articular MIA at a dose of 2 mg resulted in microgliosis within both the dorsal and ventral horn as demonstrated by immunohis- tochemistry for the microglial marker Iba1 (Figure 5A). Sham rats, which received intra-articular saline, had no microgliosis at day 7 and were indistinguishable from naives. Microgliosis produced by 2 mg MIA treatment was associated with a significant shift to effector morphometry, both dorsally and ventrally, by day 7 compared to sham (Figure 5C and D, One-way ANOVA with Dunnett’s multiple comparison test, n =467d sham, 1162 mg MIA, P,0.05). In 2 mg MIA rat ventral horn, cells were observed enwrapping motor Because the sciatic nerve branches into the tibial, peroneal and sural nerves posterior to the knee joint we examined toluidine blue stained semithin sections of these nerves (Figure S2). There was no evidence of structural degeneration, demyelination, inflammatory infiltrate or any other difference between ipsilateral and contralateral samples at any of the sites assessed at day 7 following 2 mg MIA induction. To investigate the possibility of a ‘dying back’ small fibre neuropathy, intra-epidermal nerve fibre density (IENFD) was quantified in the hindpaw. This measure is often used clinically and experimentally [27,37] to assess small fibre integrity in PLoS ONE \| www.plosone.org March 2012 \| Volume 7 \| Issue 3 \| e33730 5 Peripheral Neuropathy in the MIA Model of OA Figure 3. Approximation of total DRG ATF-3 expression. A, B, C - DRG profiles show nuclear expression of ATF-3 7 days after 2 mg (A) or 1 mg (B) MIA, but not 14 days after saline sham injection (C). D - Estimated fraction of DRG profiles expressing ATF-3 in 2 mg MIA-treated animals (One-way ANOVA with Dunnett’s multiple comparison test, n = 9614d sham, 763d, 1267d MIA, 7614d MIA, P,0.01 *, P,0.05 ). E - Comparison of ATF-3 expression in 2 mg vs 1 mg MIA injected groups at 7 day timepoint (1 way ANOVA with Bonferroni’s multiple comparison test, n = 967d sham, 1262 mg MIA, 1261 mg MIA, P,0.01 P,0.001 ). doi:10.1371/journal.pone.0033730.g003 Figure 3. Approximation of total DRG ATF-3 expression. Intra-articular MIA is associated with a reduction in ipsilateral hindpaw intra-epidermal nerve fibre density Pregabalin inhibited electrically evoked responses in the C fibre range and 8 g and 60 g evoked mechanical responses to a significantly greater extent in 2 mg MIA treated animals than in 1 mg treated animals (Figure 6A, B, Kruskal-Wallis test with Dunn’s post test, n = sham69, 1 mg MIA, 68, 2 mg MIA69). Intra-articular MIA is associated with a reduction in ipsilateral hindpaw intra-epidermal nerve fibre density in MIA animals (as above). These neurones were characterised as wide dynamic range cells (WDR), in that they gave a graded response to a wide range of innocuous and noxious mechanical and thermal stimuli, exhibited windup, and were located in laminae V–VI of the dorsal horn, as previously described [40]. As shown previously [19], pregabalin has minor inhibitory effects on evoked responses in animals that underwent an intraarticular saline sham injection 14 days before recording (Figure 6A, B and C black traces show drug effect on electrical, mechanical and thermal evoked responses, respectively). WDR cells in animals that received 2 mg MIA were significantly more sensitive to pregabalin inhibition (Figure 6A, B, C grey dotted trace). In contrast, pregabalin inhibition in animals that received 1 mg MIA was not significantly different to sham for any of the evoked responses assessed (Figure 6A, B, C grey solid vs black traces). Pregabalin inhibited electrically evoked responses in the C fibre range and 8 g and 60 g evoked mechanical responses to a significantly greater extent in 2 mg MIA treated animals than in 1 mg treated animals (Figure 6A, B, Kruskal-Wallis test with Dunn’s post test, n = sham69, 1 mg MIA, 68, 2 mg MIA69). in MIA animals (as above). These neurones were characterised as wide dynamic range cells (WDR), in that they gave a graded response to a wide range of innocuous and noxious mechanical and thermal stimuli, exhibited windup, and were located in laminae V–VI of the dorsal horn, as previously described [40]. laminae V–VI of the dorsal horn, as previously described [40]. As shown previously [19], pregabalin has minor inhibitory effects on evoked responses in animals that underwent an intraarticular saline sham injection 14 days before recording (Figure 6A, B and C black traces show drug effect on electrical, mechanical and thermal evoked responses, respectively). WDR cells in animals that received 2 mg MIA were significantly more sensitive to pregabalin inhibition (Figure 6A, B, C grey dotted trace). In contrast, pregabalin inhibition in animals that received 1 mg MIA was not significantly different to sham for any of the evoked responses assessed (Figure 6A, B, C grey solid vs black traces). Intra-articular MIA is associated with a reduction in ipsilateral hindpaw intra-epidermal nerve fibre density A, B, C - DRG profiles show nuclear expression of ATF-3 7 days after 2 mg (A) or 1 mg (B) MIA, but not 14 days after saline sham injection (C). D - Estimated fraction of DRG profiles expressing ATF-3 in 2 mg MIA-treated animals (One-way ANOVA with Dunnett’s multiple comparison test, n = 9614d sham, 763d, 1267d MIA, 7614d MIA, P,0.01 , P,0.05 ). E - Comparison of ATF-3 expression in 2 mg vs 1 mg MIA injected groups at 7 day timepoint (1 way ANOVA with Bonferroni’s multiple comparison test, n = 967d sham, 1262 mg MIA, 1261 mg MIA, P,0.01 P,0.001 ). doi:10.1371/journal.pone.0033730.g003 neuronal cell bodies. Microgliosis was noted at levels L3–L5, but was most consistent at L4, where quantification was performed. GFAP immunoreactivity was quantified in each spinal cord quadrant to assess astrogliosis, but showed no significant change (data not shown). As iba-1 reactivity peaked at the 7d timepoint, this timepoint was selected to assess 1 mg MIA group microglial activity. Following 1 mg treatment the total number of microglia counted was not significantly greater than in saline injected 7d sham animals, and was significantly less than the count in 2 mg MIA rat dorsal horn (Figure 5B). Although there was a trend for increased change in morphology in the 1 mg group, this was not significant (Figure 5E, One-way ANOVA with Bonferroni’s multiple comparison test, n = 467d sham, 1162 mg MIA, 861 mg MIA, P.0.05 1 mg vs 7d sham, P,0.05 2 mg vs 1 mg). neuronal cell bodies. Microgliosis was noted at levels L3–L5, but was most consistent at L4, where quantification was performed. GFAP immunoreactivity was quantified in each spinal cord quadrant to assess astrogliosis, but showed no significant change (data not shown). As iba-1 reactivity peaked at the 7d timepoint, this timepoint was selected to assess 1 mg MIA group microglial activity. Following 1 mg treatment the total number of microglia counted was not significantly greater than in saline injected 7d sham animals, and was significantly less than the count in 2 mg MIA rat dorsal horn (Figure 5B). Although there was a trend for increased change in morphology in the 1 mg group, this was not significant (Figure 5E, One-way ANOVA with Bonferroni’s multiple comparison test, n = 467d sham, 1162 mg MIA, 861 mg MIA, P.0.05 1 mg vs 7d sham, P,0.05 2 mg vs 1 mg). Peripheral Neuropathy in the MIA Model of OA Figure 4. Quantification of intrapepidermal nerve fibre density in plantar hindpaw following MIA treatment. A, B - sections of naı¨ve (A) and MIA (B) plantar hindpaw skin containing PGP9.5 immunoreactive intraepidermal nerve fibres (arrows). C – quantification of IENFs at the 7 and 14 day timepoints (paired t-test of ipsi vs contralateral density, n = 867d, 4614d, P,0.05). IENF density reduction is not seen in 1 mg MIA animals. doi:10.1371/journal.pone.0033730.g004 deep dorsal horn WDR cell evoked responses in 2 mg MIA treated animals, but not in 1 mg- or sham treated animals. In studying pain-related behaviour, previous investigators have used a range of doses from 1 to 4.8 mg MIA. We elected to use either 1 or 2 mg, at the lower end of this dose range. Dose- dependent effects of MIA have been demonstrated for weight- bearing asymmetry, cartilage biosynthetic processes and bone density loss [6,10]. These effects tend to plateau quickly at doses above 1 mg and indeed we found no difference in cartilage proteoglycan loss comparing 2 and 1 mg MIA; the behavioural measures of weight-bearing asymmetry and the response to acetone cooling were also not significantly different. However mechanical pain related hypersensitivity of the hindpaw was strikingly increased at the 2 mg versus 1 mg dose. We used the expression of ATF-3 [33,34] to assess the degree of injury to peripheral neurons following intra-articular MIA injection, in order to explore the possibility that differential degrees of neuronal injury may underlie the differing evoked behavioural phenotypes in the 1 mg and 2 mg MIA groups. One previous study has shown increased expression of ATF-3 in lumbar DRG cells using 1 mg MIA treatment, however these findings were not expressed in a manner in which it was possible to gauge the proportion of all DRG cells expressing ATF-3, and focussed more on timepoints after 14d [22]. There were a number of aspects of the ATF-3 response indicating that injury following intra-articular MIA is not restricted to joint afferents. The proportion of DRG cell profiles expressing ATF-3 was highest in L4 and 5, DRGs which principally project to the hindpaw, rather than L3 and 4, which contain the majority of joint afferents [35]. Peripheral Neuropathy in the MIA Model of OA The size of the ATF-3 signal peaked at ,20% of L4/L5 DRG profiles and cannot therefore have arisen solely from damage to the relatively small population (4.6%) of DRG intra-articular afferents, although it may include these afferents [36]. The lower dose of 1 mg MIA was associated with ATF-3 expression in much lower percentage of DRG cell profiless (7.6% of L4). p ( ) The signal seen in the MIA model could be the result of a direct action of MIA on the peripheral nerves running adjacent to the knee, perhaps secondary to leakage of MIA from the intra- articular space. We are highly experienced in this model and every effort is made to prevent any leakage of MIA at the time of intra- articular injection. Retrograde tracers used in MIA rat joint actually label a smaller proportion of DRG cells compared to those traced from a normal joint [36] implying that it is not the case that joint capsule disruption during the course of the model could cause MIA to diffuse to a more widespread range of tissues. We do not see any evidence of systemic effects of MIA and the increased ATF-3 expression was always strictly unilateral, as were other markers of neuronal injury discussed below. MIA is able to induce massive Ca2+ influx in rat peripheral nerve in vitro via inhibition of metabolism, as well as being cytotoxic to non- neuronal cells such as astrocytes [41,42]. Figure 4. Quantification of intrapepidermal nerve fibre density in plantar hindpaw following MIA treatment. A, B - sections of naı¨ve (A) and MIA (B) plantar hindpaw skin containing PGP9.5 immunoreactive intraepidermal nerve fibres (arrows). C – quantification of IENFs at the 7 and 14 day timepoints (paired t-test of ipsi vs contralateral density, n = 867d, 4614d, P,0.05). IENF density reduction is not seen in 1 mg MIA animals. doi:10.1371/journal.pone.0033730.g004 Systemic Pregabalin Reduces Deep Dorsal Horn Evoked Responses in 2 mg, but not 1 mg MIA treated animals Systemic Pregabalin Reduces Deep Dorsal Horn Evoked Responses in 2 mg, but not 1 mg MIA treated animals Systemic Pregabalin Reduces Deep Dorsal Horn Evoked Responses in 2 mg, but not 1 mg MIA treated animals Systemic Pregabalin Reduces Deep Dorsal Horn Evoked Responses in 2 mg, but not 1 mg MIA treated animals Extracellular action potentials were recorded from deep dorsal horn neurones with receptive fields on the plantar hindpaw, where behavioural hyperresponse to normally innocuous stimuli is seen Extracellular action potentials were recorded from deep dorsal horn neurones with receptive fields on the plantar hindpaw, where behavioural hyperresponse to normally innocuous stimuli is seen PLoS ONE \| www.plosone.org March 2012 \| Volume 7 \| Issue 3 \| e33730 6 Peripheral Neuropathy in the MIA Model of OA PLoS ONE \| www.plosone.org Discussion In this study we have carefully characterised the widely used MIA model of OA in relation to the development of pain related behaviour, the degree of joint cartilage loss and neural injury, and the extent of dorsal horn pharmacological plasticity. To summarise, the higher dose of MIA (2 mg) was associated with greater hindpaw mechanical hypersensitivity than the lower dose (1 mg) treatment. However there was no difference in the degree of cartilage proteoglycan loss. 2 mg, but not 1 mg, MIA produced an increase in the expression of the injury marker ATF-3 in DRG cells, a reduction in intra-epidermal nerve fibre density within plantar epidermis, and ipsilateral spinal cord microgliosis, indicating significant neural injury. Systemic pregabalin significantly inhibited Intra-articular MIA triggers a vigorous inflammatory response up to day 5 post-injection, with associated monocyte and neutrophil infiltration and joint swelling [5,9]. A similar degree of joint swelling is observed in other inflammatory models of monoarthritis, meaning that swelling alone is unlikely to account for the greater ATF-3 signal observed in the MIA model [45]. It has recently been shown that some algogens, such as formalin and capsaicin, which strongly activate subsets of primary afferents when injected into the plantar hindpaw, trigger ATF-3 expression in DRG cells [43]. This raises the point as to whether inflammatory mediators released as a consequence of intra- articular MIA could contribute to increased ATF-3 expression. March 2012 \| Volume 7 \| Issue 3 \| e33730 March 2012 \| Volume 7 \| Issue 3 \| e33730 PLoS ONE \| www.plosone.org 7 Peripheral Neuropathy in the MIA Model of OA Figure 5. Quantification of microglial activation in spinal cord following MIA treatment. A, B - expression of the microglial marker iba1 in L4 spinal cord in 2 mg (A) and 1 mg (B) MIA groups. C, D – Quantification of microglia with effector morphology in dorsal (C) and ventral (D) horn (1- way ANOVA, n = 467d sham, 1162 mg MIA, P,0.05 ). E – Comparison of dorsal horn microgliosis in 2 mg vs 1 mg MIA groups at the 7d timepoint (1-way ANOVA, n = 467d sham, 1162 mg MIA, 861 mg MIA, P,0.05 ). doi:10.1371/journal.pone.0033730.g005 Figure 5. Quantification of microglial activation in spinal cord following MIA treatment. A, B - expression of the microglial marker iba1 in L4 spinal cord in 2 mg (A) and 1 mg (B) MIA groups. Discussion C, D – Quantification of microglia with effector morphology in dorsal (C) and ventral (D) horn (1- way ANOVA, n = 467d sham, 1162 mg MIA, P,0.05 ). E – Comparison of dorsal horn microgliosis in 2 mg vs 1 mg MIA groups at the 7d timepoint (1-way ANOVA, n = 467d sham, 1162 mg MIA, 861 mg MIA, P,0.05 ). doi:10.1371/journal.pone.0033730.g005 Additionally, although inflammatory measures resolve by day 7 in the MIA model [10] ATF-3 signal is still present at day 14. This would appear to rule out a link between ATF-3 signal and inflammation. A further assessment of ATF-3 expression, a well as the other measures used in this study, at later timepoints such as 28 days after injection, would illuminate whether the pathological ATF-3 expression has now been assessed in a number of different models of inflammation all of which are associated with enhanced pain-related behaviour. But neither intra-plantar application of Complete Freund’s Adjuvant, antigen induced arthritis nor collagen-induced arthritis could induce ATF-3 expression above the level of ,2% in lumbar DRG [44,45]. Figure 6. Effect of pregabalin treatment (10 mg/kg s.c.) on evoked responses of deep dorsal horn WDR neurones. A - Electrically evoked responses in the Aß, Ah and C fibre range, as well as PD (post-discharge/repetitive firing) expressed as % of predose baseline in 2 mg MIA, 1 mg MIA or sham injected animals. B - Mechanically evoked responses expressed as % of predose baseline in 2 mg MIA, 1 mg MIA or sham injected animals. C - Thermally evoked responses expressed as % of predose baseline in 2 mg MIA, 1 mg MIA or sham injected animals. Note the significantly greater effectiveness of pregabalin in the 2 mg treated group (normalized post-drug responses compared using Kruskal Wallis test with Dunn’s posttest. n = 14d sham69, 1 mg MIA68, 2 mg MIA69. P,0.05 , P,0.01, *). The sham and 2 mg data is included for comparison, but has previously been published in a different form [19]. doi:10.1371/journal.pone.0033730.g006 Figure 6. Effect of pregabalin treatment (10 mg/kg s.c.) on evoked responses of deep dorsal horn WDR neurones. A - Electrically evoked responses in the Aß, Ah and C fibre range, as well as PD (post-discharge/repetitive firing) expressed as % of predose baseline in 2 mg MIA, 1 mg MIA or sham injected animals. Peripheral Neuropathy in the MIA Model of OA Peripheral Neuropathy in the MIA Model of OA changes observed here persist, exacerbate or reverse with time. However, the existence of a robust non-inflammatory pain behavioural phenotype at the 14d timepoint, which neither exacerbates nor resolves over succeeding weeks [5], as well as the well characterised spinal pharmacological plasticity at this stage [18,19] justify the focus of the present study on this timepoint. or sham treated animals, may thus indicate the presence of central sensitization in the 2 mg group that is not present in the 1 mg animals. This may account for the reduced behavioural hypersen- sitivity to stimuli applied to the hindpaw referred-pain area in 1 mg MIA treated animals compared to more sensitized 2 mg treated group. Alternatively, the differential effect of pregabalin could reflect the differing extents of peripheral neuronal injury in the 2 mg and 1 mg groups. Because of the strong ATF-3 signal in response to MIA in L4 and 5 DRG we studied the effects of this treatment on the innervation of hindpaw skin, a major peripheral target of these neurones. Quantification of intra-epidermal nerve fibre density (IENFD) is a sensitive means of assessing injury to small fibres used in both clinical practice [37] and animal models of toxic neuropathies [46,47,48,49]. 14 days after intra-articular 2 mg MIA treatment, there was a 38% reduction in IENF density of hindpaw skin. In contrast, treatment with 1 mg MIA did not reduce IENFD. It should be noted that while the presence of central sensitization is often found to be a necessary condition for gabapentinoid efficacy, it is clearly not a sufficient condition. Hence, although central sensitization may be present in some other inflammatory chronic pain states, in the absence of additional factors (such as the presence of neuropathy, or activation of descending serotonergic facilitatory controls) gaba- pentinoid efficacy does not increase [19,65]. Clinical studies suggest the existence of a subpopulation of OA patients who experience pain with neuropathic features. In patients undergoing surgical management of OA, pain often persists after joint replacement [23]. A recent imaging study reveals abnormal descending controls from brain to spinal cord in a subgroup of patients with OA [24], an event seen in animal neuropathic models as well as in the MIA model [19,25], with the extent of this change correlating with patients’ use of neuropathic pain descriptors. Peripheral Neuropathy in the MIA Model of OA Coupled with the demonstration of increased central sensitization and referred pain [67], these studies suggest the existence of central changes in OA resembling those seen in neuropathic pain. This reduction in IENFD is similar to that previously reported in models of painful toxic neuropathies, for instance the 24–44% reduction seen in paclitaxel- and vincristine- experimental painful neuropathies [46]. There are broad similarities in the behavioural and anatomical phenotypes in 2 mg intra-articular MIA and experimental paclitaxel-neuropathy. Both share hindpaw mechan- ical and cooling, but not thermal, hypersensitivity, despite IENF loss, features common to many painful neuropathies [48,50]. Traumatic nerve injury models are associated with a response in dorsal horn microglia, accompanied by subsequent astrocytosis, which may contribute to the development and maintenance of neuropathic pain [51,52,53]. In response to injury, microglia proliferate [29,54,55], migrate to the site of injury [56,57] and undergo a morphological change to become less ramified and more amoeboid [58]. In terms of function, they can phagocytose cellular debris [59], present antigens [60] and secrete a broad range of cytokines and chemokines which amplify the transmission of nociceptive information in the dorsal horn [51]. The induction of microgliosis appears dependent on primary afferent electrical activity, as well as numerous neuron-to-glia signalling molecules including neuregulin-1, CCL2 and fractalkine [29] Furthermore, in addition to reported cutaneous hypoaesthesia with paradoxical mechanical allodynia overlying the degenerating knee in OA patients, there is also loss of peripheral vibratory sense distal to the degenerating knee in these patients, with the degree of loss of acuity proportional to the radiographic OA severity [68,69,70]. Whether this loss correlates to any alteration in IENF in these distal territories clinically is currently unknown. y y There is considerable variation in the dose and volume used preclinically to induce the MIA model. Assessment of 43 studies published to date indicates that 1, 2 and 3 mg MIA doses were in equally common usage (Table S2). The available data suggests that there are different pathophysiological sequellae that follow different doses of MIA, with the 2 mg dose associated with a much greater degree of neuronal injury and/or central sensitiza- tion than 1 mg MIA. This includes a ‘dying back’ of small fibre afferents innervating targets outside of the knee joint, which, if it can be attributed to neurotoxicity, that would call into question the utility of the 2 mg MIA model for translational study of osteoarthritis pain. Peripheral Neuropathy in the MIA Model of OA However, the concurrent neuropathic and osteoarthritic phenotypes of the 2 mg MIA model, irrespective of the etiology of the former, could still allow the use of this model to investigate clinical osteoarthritic pain with neuropathic features/ descriptors Following 2 mg intra-articular MIA, we noted increased numbers of ‘effector’ microglia within both the dorsal and ventral horn. The 1 mg MIA treatment did not increase the total number of microglia within the dorsal horn nor lead to a significant change in their morphology. Previous studies have shown that the microglial response within the dorsal horn is robustly evoked by traumatic nerve injury but that certain models of chemotherapy- evoked peripheral neuropathy [61] and most inflammatory pain models [62,63] do not produce a microglial response, with some exceptions [64]. This suggests that there may be a threshold of stress or axonal injury that a neuron needs to achieve before microglia can be recruited into a pro-inflammatory response, even when a ‘dying back’ neuropathy of sensory neurones is present. The presence of microglial activation at the 14d timepoint, while inflammation resolves at the 7d timepoint [9], also suggests that microglial activation in this case is not primarily linked to inflammation In summary, the divergent sequellae following different doses of MIA must be taken into account when interpreting data generated using the model, especially where indirect outcome measures of joint pain have been employed and where doses greater than 1 mg MIA are used. Numerous studies indicate that gabapentinoid drugs, such as pregabalin, are significantly better able to modulate spinal innocuous and nociceptive transmission in models of neuropathy or other pain states than under physiological conditions [19,65,66]. This is supported clinically by functional magnetic resonance imaging evidence that gabapentin is significantly better able to modulate nociceptive transmission following the induction of central sensitiza- tion [21]. The ability of pregabalin to reduce deep dorsal horn evoked responses significantly in the 2 mg MIA group, but not in 1 mg MIA PLoS ONE \| www.plosone.org Discussion B - Mechanically evoked responses expressed as % of predose baseline in 2 mg MIA, 1 mg MIA or sham injected animals. C - Thermally evoked responses expressed as % of predose baseline in 2 mg MIA, 1 mg MIA or sham injected animals. Note the significantly greater effectiveness of pregabalin in the 2 mg treated group (normalized post-drug responses compared using Kruskal Wallis test with Dunn’s posttest. n = 14d sham69, 1 mg MIA68, 2 mg MIA69. P,0.05 , P,0.01, **). The sham and 2 mg data is included for comparison, but has previously been published in a different form [19]. doi:10.1371/journal.pone.0033730.g006 March 2012 \| Volume 7 \| Issue 3 \| e33730 8 Peripheral Neuropathy in the MIA Model of OA March 2012 \| Volume 7 \| Issue 3 \| e33730 References (2009) Psychophysical and functional imaging evidence supporting the presence of central sensitization in a cohort of osteoarthritis patients. Arthritis Rheum 61: 1226–1234. 8. Combe R, Bramwell S, Field MJ (2004) The monosodium iodoacetate model of osteoarthritis: a model of chronic nociceptive pain in rats? Neuroscience Letters 370: 236–240. 25. Bee LA, Dickenson AH (2007) Rostral ventromedial medulla control of spinal sensory processing in normal and pathophysiological states. Neuroscience 147: 786–793. 9. Clements KM, Ball AD, Jones HB, Brinckmann S, Read SJ, et al. (2009) Cellular and histopathological changes in the infrapatellar fat pad in the monoiodoacetate model of osteoarthritis pain. 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Acknowledgments The authors are grateful to Ning Zhu, Rose Fricker, Margarita Calvo, Leonor Gonc¸alves and Shafaq Sikandar for technical assistance. Matthew Thakur, Anthony H. Dickenson and David L. H. Bennett are members of the London Pain Consortium. The authors are grateful to Ning Zhu, Rose Fricker, Margarita Calvo, Leonor Gonc¸alves and Shafaq Sikandar for technical assistance. Matthew Thakur, Anthony H. Dickenson and David L. H. Bennett are members of the London Pain Consortium. Table S1 Mean cartilage proteoglycan score for 2 mg MIA, 1 mg MIA and saline injected joints, as assessed using toluidine blue staining (See Fig. 1C, D.). Scoring reflects the degree of proteoglycan loss, with 12 corresponding to total loss over the entirety of both condylar surfaces, while 1 is undisrupted cartilage proteoglycan. n = 461 mg, 362 mg ani- mals, +14d saline injected contralateral controls. Supporting Information Figure S1 Spinal cord expression of ATF-3 following 2 mg MIA treatment. A - nuclear expression of ATF-3 in magnocellular ventral horn ipsilateral to 2 mg MIA injection. B - quantification of total ATF-3+ nuclei/4 sections counted (one-way ANOVA with Dunnett’s multiple comparison test, n = 8 animals PLoS ONE \| www.plosone.org March 2012 \| Volume 7 \| Issue 3 \| e33730 9 Peripheral Neuropathy in the MIA Model of OA per timepoint, p,0.05). 1 mg MIA was not associated with ventral horn ATF-3 expression at day 7. (TIF) Table S2 43 published studies which used the MIA model. Columns denote dose of monoiodoacetate used. The highest dose was 4.8 mg, the lowest was 0.01 mg. Studies in bold specifically assessed evoked pain responses at the hindpaw. Where multiple doses were used, a study is referenced at each dose. (DOC) Table S2 43 published studies which used the MIA model. Columns denote dose of monoiodoacetate used. The highest dose was 4.8 mg, the lowest was 0.01 mg. Studies in bold specifically assessed evoked pain responses at the hindpaw. Where multiple doses were used, a study is referenced at each dose. (DOC) Figure S2 Analysis of peripheral nerve following 2 mg MIA injection. A, B - toluidine-blue stained semithin sections of common peroneal nerve ipsilateral (A) or contralateral (B) to 2 mg MIA injection at day 7 of the model. There is no visible axon degeneration, demyelination or inflammatory cell infiltrate (n = 4 per group). (TIF) Author Contributions Conceived and designed the experiments: MT AD DB. Performed the experiments: MT WR. Analyzed the data: MT. Contributed reagents/ materials/analysis tools: CH. Wrote the paper: MT. Conceived and designed the experiments: MT AD DB. Performed the experiments: MT WR. Analyzed the data: MT. Contributed reagents/ materials/analysis tools: CH. Wrote the paper: MT. 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PLoS ONE \| www.plosone.org March 2012 \| Volume 7 \| Issue 3 \| e33730 March 2012 \| Volume 7 \| Issue 3 \| e33730 10 Peripheral Neuropathy in the MIA Model of OA Peripheral Neuropathy in the MIA Model of OA Diabetes Care 27: 1974. 67. Iannetti GD, Zambreanu L, Wise RG, Buchanan TJ, Huggins JP, et al. (2005) Pharmacological modulation of pain-related brain activity during normal and central sensitization states in humans. Proc Natl Acad Sci USA 102: 18195–18200. 50. Peters CM, Jimenez-Andrade JM, Jonas BM, Sevcik MA, Koewler NJ, et al. (2007) Intravenous paclitaxel administration in the rat induces a peripheral sensory neuropathy characterized by macrophage infiltration and injury to sensory neurons and their supporting cells. Experimental Neurology 203: 42–54. 68. Arendt-Nielsen L, Nie H, Laursen MB, Laursen BS, Madeleine P, et al. (2010) Sensitization in patients with painful knee osteoarthritis. Pain 149: 573–581. 69. Hendiani JA, Westlund KN, Lawand N, Goel N, Lisse J, et al. 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https://openalex.org/W4323927571	https://zenodo.org/records/7720352/files/A_esquizofrenia_das_vozes_do_mercado_das_maos_invisiveis.pdf	Portuguese	null	A esquizofrenia das "vozes do mercado" e das "mãos invisíveis" contra as vozes e as mãos dos trabalhadores (The schizophrenia of "market voices" and "invisible hands" against the voices and hands of workers)	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	1,053	A esquizofrenia das “vozes do mercado” e das “mãos invisíveis” contra as vozes e as mãos dos trabalhadores Edmar Roberto Prandini Gestor Governamental edmarrp@yahoo.com.br Março de 2023 Nos meses recentemente passados, uma grande empresa varejista brasileira sofreu um abalo em sua história em função de “inconsistências contábeis” que se refletiram, como se sabe, na exposição de um altíssimo endividamento, na casa de várias dezenas de bilhões de reais. No contexto de uma forte concorrência com outras empresas varejistas nacionais e com o varejo eletrônico, inclusive da China, somado ao encarecimento do crédito, em função da acelerada elevação da taxa Selic, que reverbera sobre todos os créditos e financiamentos no país, a empresa viu-se forçada a recorrer à “recuperação judicial” para evitar ser destruída pela ação de alguns grandes bancos que lhe haviam fornecido linhas de crédito também bilionárias, agora lançadas como provisionamento (possíveis prejuízos) para evitar contaminar os balanços e os resultados destas próprias instituições bancárias. Ressalte-se que a empresa varejista possui outros milhares de credores com saldos a receber menores do que os dos bancos, mas que, somados, também causam estrago na capacidade da empresa funcionar, se forçada a antecipar os pagamentos ou se impedida de renovar os créditos para manter-se girando suas engrenagens. Surpreendeu a todos o fato de que as agências de classificação de risco e de auditoria não tivessem indicado anteriormente a existência dessas tais “inconsistências contábeis”. Repetiu-se, com tais agências de risco e de auditoria, de modo mais localizado e com menor irradiação, o mesmo que aconteceu na crise americana de 2008-2009, que afetou intensamente a economia de todo o mundo? Por sua vez, o Banco Central (independente, como queria o “mercado”) também foi obrigado a vir a público para informar que os seus resultados precisavam ser corrigidos, porque uma contabilização errônea de valores havia provocado uma divulgação aparentemente mais positiva de valores que efetivamente não haviam se realizado. Finalmente, há poucos dias, a divulgação dos números do PIB 2022 demonstraram que a economia brasileira veio desacelerando durante o ano de 2022, voltando ao negativo no ultimo trimestre do ano, apesar dos volumosos e extraordinários estímulos financeiros do governo Bolsonaro, aprovados no Congresso às vésperas das eleições, em sua tentativa de impedir a vitória eleitoral de Lula, o que, obviamente, não se logrou realizar. A esquizofrenia das “vozes do mercado” e das “mãos invisíveis” contra as vozes e as mãos dos trabalhadores Todos estes elementos somados complementam-se com algumas vozes do “mercado” anunciando que aparentemente o país está às vésperas de uma crise de crédito, que tenderia a emperrar toda a economia, ao mesmo tempo em que, dizem, o governo Lula 3 (2023-2026) estaria orientando-se para rumos “heterodoxos” moldados por uma perspectiva de “Estado indutor” que seria, segundo aquelas “vozes do mercado”, um percurso errado, sufocando a “liberdade” dos “agentes econômicos”. Desde a publicação do clássico de Adam Smith, A Riqueza das Nações, as tais demiúrgicas “mãos invisíveis” que seriam capazes de produzir “riqueza para as nações” não se entenderam com nenhuma proposta de igualdade social e econômica, nem dentro de nenhuma nação nem nas relações internacionais. Apesar do acelerado incremento da produção mundial, movido pelas revoluções decorrentes das inovações tecnológicas, as “mãos invisíveis” sempre alinharam-se a “olhos cegos” e a “decisões injustas” e produziram concentração alarmante de riquezas, favorecendo alguns, enquanto pobreza para uma amplíssima maioria e devastação para grandes regiões do planeta. Será, pois, que foram estas mesmas “mãos invisíveis”, estes mesmos “olhos cegos”, estas “decisões injustas” e estas mesmas tais “vozes do mercado” que contrataram as mesmas agências de classificação de risco e as mesmas empresas de auditoria que continuaram errando as contas por “inconsistências contábeis” que resultam em fraudes, prejuízos e crise social e ambiental planetária? Confesso que, ao contrário dessa esquizofrenia de “mãos invisíveis” e de “vozes de mercado”, prefiro o que acusam de ser arcaísmo: as representações dos trabalhadores organizados em associações e sindicatos, reivindicando direitos trabalhistas, por exemplo; prefiro as passeatas democráticas ao invés dos “acampamentos bolsonaristas” clamando por intervenção militar e destruindo as sedes dos poderes em Brasília; prefiro a estabilidade do servidor público, inclusive da Receita Federal na alfândega, por exemplo, ao invés da “reforma administrativa” que amedrontará quem resista a deixar entrarem “presentes”, “jóias” e “colares” para o “acervo” particular da “primeira dama”, contra todas as normativas abandonadas se o “cliente” for “poderoso”. É curiosa a inversão na narrativa histórica dessa linguagem “esquizofrênica” do “liberalismo” das “mãos invisíveis” e das “vozes do mercado”: enquanto esses preceitos dogmáticos e ortodoxos do “capitalismo liberal” que defendem a “livre flutuação dos preços” e da “moeda” tiveram sua certidão de nascimento datada do século XVIII, os “arcaísmos” do discurso dos direitos de cidadania, dos direitos humanos, dos direitos dos trabalhadores, dos direitos sociais e dos direitos ambientais, nasceram nos séculos XIX, XX e neste século. A esquizofrenia das “vozes do mercado” e das “mãos invisíveis” contra as vozes e as mãos dos trabalhadores Aquela “esquizofrenia” nasceu antes, mas a chamam de “inovação”, “novidade”, “liberdade econômica”, enquanto o discurso dos direitos, que se articulou depois, é chamado de “velho” e “antiquado”. Vejam como a luta contra o racismo e contra o machismo continuam a ser novíssimas, com poucas décadas de nascimento como direitos no Brasil, nos Estados Unidos, na África do Sul ou na Índia. Este é o contrassenso dos que querem opor-se à discussão das taxas de juros Selic que o novíssimo Governo Lula 2023-2026 está fazendo. Baixar os juros, melhorar os salários, estimular o crescimento do PIB nas áreas mais intensivas de mão de obra, para estimular o crescimento do emprego, ampliar as políticas sociais e as estratégias de redução das desigualdades, inclusive pela reforma do sistema de tributos sobre o consumo e a renda são a verdadeira economia política de que precisamos. As vozes do mercado que devem ser ouvidas são as vozes dos pobres e não das agências de classificação de risco. Que 2023 entre na história como o ano em que ao menos a reforma tributária do consumo introduzindo um único sistema de IVA (imposto sobre o valor agregado), com uma única alíquota, seja aprovado em emenda constitucional, melhorando profundamente as condições de operacionalização cotidiana da atividade econômica no páis inteiro, aumentando a transparência do sistema tributário e reduzindo as fraudes de “inconsistências contábeis” que, na maioria das vezes, acabam servindo muito bem aos “gatunos” das “mãos invisíveis”.
https://openalex.org/W2981043513	https://www.duo.uio.no/bitstream/10852/92430/1/Structural%2bbrain%2bimaging%2bstudies%2boffer%2bclues%2babout%2bthe%2beffects%2bof%2bthe%2bshared%2bgenetic%2betiology%2bamong%2bneuropsychiatric%2bdisorders.pdf	English	null	Structural Brain Imaging Studies Offer Clues about the Effects of the Shared Genetic Etiology among Neuropsychiatric Disorders	bioRxiv (Cold Spring Harbor Laboratory)	2,019	cc-by	8,917	Abstract Genomewide association studies have found signiﬁcant genetic correlations among many neuropsychiatric disorders. In contrast, we know much less about the degree to which structural brain alterations are similar among disorders and, if so, the degree to which such similarities have a genetic etiology. From the Enhancing Neuroimaging Genetics through Meta- Analysis (ENIGMA) consortium, we acquired standardized mean differences (SMDs) in regional brain volume and cortical thickness between cases and controls. We had data on 41 brain regions for: attention-deﬁcit/hyperactivity disorder (ADHD), autism spectrum disorder (ASD), bipolar disorder (BD), epilepsy, major depressive disorder (MDD), obsessive compulsive disorder (OCD), and schizophrenia (SCZ). These data had been derived from 24,360 patients and 37,425 controls. The SMDs were signiﬁcantly correlated between SCZ and BD, OCD, MDD, and ASD. MDD was positively correlated with BD and OCD. BD was positively correlated with OCD and negatively correlated with ADHD. These pairwise correlations among disorders were correlated with the corresponding pairwise correlations among disorders derived from genomewide association studies (r = 0.494). Our results show substantial similarities in sMRI phenotypes among neuropsychiatric disorders and suggest that these similarities are accounted for, in part, by corresponding similarities in common genetic variant architectures. Structural brain imaging studies offer clues about the effects of the shared genetic etiology among neuropsychiatric disorders Nevena V. Radonjić 1 ●Jonathan L. Hess 2 ●Paula Rovira 3,4 ●Ole Andreassen5 ●Jan K. Buitelaar 6,7,8 ● Christopher R. K. Ching9 ●Barbara Franke 7,10,11 ●Martine Hoogman10 ●Neda Jahanshad 12 ●Carrie McDonald13 ● Lianne Schmaal 14,15 ●Sanjay M. Sisodiya16,17 ●Dan J. Stein 18 ●Odile A. van den Heuvel19 ● Theo G. M. van Erp 20,21 ●Daan van Rooij22 ●Dick J. Veltman19 ●Paul Thompson23 ●Stephen V. Faraone 2 Received: 29 May 2020 / Revised: 7 December 2020 / Accepted: 11 December 2020 / Published online: 17 January 2021 © Th A h ( ) 2021 Thi i l i bli h d i h Nevena V. Radonjić 1 ●Jonathan L. Hess 2 ●Paula Rovira 3,4 ●Ole Andreassen5 ●Jan K. Buitelaar 6,7,8 ● Christopher R. K. Ching9 ●Barbara Franke 7,10,11 ●Martine Hoogman10 ●Neda Jahanshad 12 ●Carrie McDonald13 ● Lianne Schmaal 14,15 ●Sanjay M. Sisodiya16,17 ●Dan J. Stein 18 ●Odile A. van den Heuvel19 ● Theo G. M. van Erp 20,21 ●Daan van Rooij22 ●Dick J. Veltman19 ●Paul Thompson23 ●Stephen V. Faraone 2 Received: 29 May 2020 / Revised: 7 December 2020 / Accepted: 11 December 2020 / Published online: 17 January 2021 © The Author(s) 2021. This article is published with open access Molecular Psychiatry (2021) 26:2101–2110 https://doi.org/10.1038/s41380-020-01002-z Molecular Psychiatry (2021) 26:2101–2110 https://doi.org/10.1038/s41380-020-01002-z ARTICLE ARTICLE Collection of structural neuroimaging summary statistics Summary statistics from ENIGMA structural neuroimaging studies were collected from 12 multisite analyses published by the ENIGMA Consortium for the following neu- ropsychiatric disorders: ADHD [10, 11], ASD [12], BD [13, 14], epilepsy [15], MDD [16, 17], OCD [18, 19], and SCZ [20, 21]. Prior to computing the summary statistics, the regional brain volumes had been segmented with a common ENIGMA protocol using FreeSurfer software. Each site performed these segmentations on their raw data. In addi- tion, quality control protocols provided by ENIGMA were run at each site. Details are at: http://enigma.ini.usc.edu/ protocols/imaging-protocols. To derive an estimate of the degree to which sMRI phenotypes were similar among disorders, we computed pairwise Spearman’s rank correlation between the Cohen’s d SMDs for each pair of disorders. We then used Pear- son’s correlation to estimate, whether the genetic corre- lations for each disorder covaried with the sMRI phenotype correlations. We used a traditional permutation framework to generate a null distribution of sMRI phe- notype correlations by randomly shufﬂing Cohen’s d values 10,000 times for each pair of disorders, then recalculating sMRI correlations from the shufﬂed sets. From the null distributions, we derived an empirical per- mutation p value for each sMRI phenotype correlation. However, a reliable p value could not be calculated due to nonindependence between pairwise caused by sample overlap between imaging studies. Adjustments for sample overlap would be possible with individual-level data, but the present study only had access to summary statistics. In a leave-one-out analysis, we iteratively excluded one pair of disorder correlations from the set and recalculated Spearman’s correlation coefﬁcients to determine whether correlations were driven by any pair of disorders. Bino- mial sign tests were used to determine whether the number of disorders showing the same direction of effect in the sMRI phenotypes was greater than expected by chance (null probability of 50%). Per brain region, we performed Cochran’s Q test implemented in the R package metafor (v.2.1–0) to determine whether variability among Cohen’s d values was greater than expected by chance. All statis- tical analyses were performed with R version 3.5.2 The ADHD and ASD samples comprised both youth and adults. The other samples comprised adults only. The eth- nicity of the patients was not available for all participants. The “epilepsy” cohort comprised temporal lobe epilepsy, genetic generalized epilepsy, and extra temporal epilepsy. Collection of structural neuroimaging summary statistics We analyzed 7 subcortical and 34 cortical regions (total of 41 brain regions; the mean of left and right structures) that were included in the above speciﬁed ENIGMA studies. We extracted the covariate-adjusted Cohen’s d standardized mean differences (SMDs) denoting the case versus unaf- fected comparison subject differences in subcortical volume and cortical thickness/surface area measures. The covariates used in these studies adjusted SMDs for several covariates as indicated in Supplementary Table 1. Introduction Note, the full meta-analysis GWAS of MDD that included data from 23andMe was not available for public release, thus we used the meta-analysis that combined results from the PGC cohorts and UK Biobank. common epilepsy syndromes [15], major depressive dis- order (MDD) [16, 17], obsessive compulsive disorder (OCD) [18, 19], and schizophrenia (SCZ) [20, 21]. Here we estimate the degree of similarity in sMRI phenotypes among these disorders and evaluate whether these similarities are inﬂuenced by corresponding similarities in common genetic variant architectures. Methods Linkage disequilibrium (LD)-score regression, a popular approach designed to analyze summary statistics from GWAS, was used to quantify the amount of shared genetic heritability, or genetic correlation (rg), existing between pairs of neuropsychiatric disorders, considering HapMap3 LD-scores [22]. For these analyses, the largest and latest GWAS available for each neuropsychiatric disorder was selected and ﬁltered to exclude markers with INFO < 0.90 or within the MHC region (hg19:chr6:25–35 Mb) (Supple- mentary Table 1). GWAS summary statistics were merged with the HapMap3 reference panel (hg37 build), wherein variants with a MAF ≥5% in the HapMap3 dataset were retained, prior to computing (co)heritability estimates. Introduction Subcortical brain volumes and cortical thickness/surface area dynamically change from early development through adulthood and old age. A study of the Enhancing Neuroi- maging Genetics through Meta-Analysis (ENIGMA) Plasti- city Working Group reported that changes in structural magnetic resonance imaging (sMRI) phenotypes have herit- abilities ranging from 5% for pallidum to 42% for cerebellar gray matter [7]. Heritability estimates of change rates were age-related and generally higher in adults than in children, probably due to an increasing inﬂuence of genetic factors with age [7]. However, it appears that later in adulthood heritability decreases most likely due to cumulative effect of environ- mental inﬂuences over the lifespan [8]. ENIGMA sMRI stu- dies of different psychiatric and neurological disorders further characterized MRI-derived phenotypes that can be used to assess heritability (reviewed in [9]). Neuropsychiatric disorders have substantial heritability, as shown by many studies of twins and families [1]. Geno- mewide association studies (GWAS) have shown that common genetic variants account for some of this herit- ability, and that some of this heritability is shared across neuropsychiatric disorders [2–5]. The genetic overlap across disorders may partly explain why these disorders tend to co- occur with one another in both clinical and community samples [6]. * Stephen V. Faraone sfaraone@childpsychresearch.org Extended author information available on the last page of the article Supplementary information The online version of this article (https:// doi.org/10.1038/s41380-020-01002-z) contains supplementary material, which is available to authorized users. * Stephen V. Faraone sfaraone@childpsychresearch.org Extended author information available on the last page of the article Supplementary information The online version of this article (https:// doi.org/10.1038/s41380-020-01002-z) contains supplementary material, which is available to authorized users. Supplementary information The online version of this article (https:// doi.org/10.1038/s41380-020-01002-z) contains supplementary material, which is available to authorized users. Supplementary information The online version of this article (https:// doi.org/10.1038/s41380-020-01002-z) contains supplementary material, which is available to authorized users. ENIGMA has also reported signiﬁcant case vs. control differences in sMRI phenotypes for: attention-deﬁcit/ hyperactivity disorder (ADHD) [10, 11], autism spectrum disorder (ASD) [12], bipolar disorder (BD) [13, 14], Extended author information available on the last page of the article 2102 N. V. Radonjić et al. Table 2 are the numbers of affected cases and unaffected control participants included in each GWAS. Results Sample demographics for the twelve studies by the ENIGMA Consortium on structural brain abnormalities in neuropsychiatric disorders are presented in Table 1. Collection of GWAS results among neuropsychiatric disorders Eighteen sMRI phenotypes exhibited homogeneity with respect to sign of Cohen’s d across each of the neuropsychiatric disorders evaluated (binomial sign test p values < 0.05): cortical thicknesses for caudal middle frontal gyrus, entorhinal cortex, fusiform gyrus, inferior temporal gyrus, insula, lateral orbitofrontal cortex, lingual gyrus, middle temporal gyrus, paracentral lobule, para- hippocampal gyrus, pars opercularis of inferior temporal gyrus, precentral gyrus, precuneus, rostral anterior cingulate cortex, and supramarginal gyrus; subcortical volume for the hippocampus; and surface area for middle temporal gyrus, pars triangularis of inferior temporal gyrus, and pericalcar- ine cortex. For sMRI phenotypes for 39 regions of interest varying degrees of heterogeneity were noted in terms of discrepancy of signs of Cohen’s d. For example, individuals with ASD showed a slightly thicker cortex in the rostral (R Core Team, 2018). We adjusted for repeated correla- tion tests using the Bonferroni procedure. Correlations showing a Bonferroni-adjusted p < 0.05 were considered signiﬁcant (threshold p = 0.00227). Collection of GWAS results among neuropsychiatric disorders Publicly available summary statistics from GWAS were downloaded from the Psychiatric Genomics Consortium (PCG) website (https://www.med.unc.edu/pgc/results-and- downloads/) with the exception of GWAS results for MDD coming from an online resource hosted by the University of Edinburgh (https://doi.org/10.7488/ds/2458) and of GWAS results for epilepsy coming from the online Epilepsy Genetic Association Database (epiGAD) (http://www.epigad.org/ gwas_ilae2018_16loci.html). Presented in Supplementary Structural brain imaging studies offer clues about the effects of the shared genetic etiology among. . . 2103 Table 1 Sample demographics for the twelve studies by the ENIGMA Consortium into structural brain alterations in neuropsychiatric disorders. Disorder MRI measure Cases (n) Controls (n) Total n Sites Weighted mean age (cases) Weighted mean age (controls) References ADHD Cortical thickness 2246 1934 4180 36 19.2 18.1 [2, 28] Surface area 2246 1934 4180 36 19.2 18.1 Subcortical volume 1713 1529 3242 23 18.6 ASD Cortical thickness 1571 1651 3222 49 15.4 [18] Surface area Subcortical volume BD Cortical thickness 1837 2582 4419 28 38.4a 35.6a [19, 30] Surface area 1820 2582 4402 28 38.4a 35.6a Subcortical volume 1710 2594 4304 20 40.1a 36.5a Epilepsy Cortical thickness 2149 1727 3876 24 34.4 33.3 [7] Surface area Subcortical volume MDD Cortical thickness 1911 7663 9574 20 44.8a 54.6a [6, 22] Surface area 1902 7658 9560 20 44.8a 54.6a Subcortical volume 1728 7199 8927 15 43.3a 56a OCD Cortical thickness 1498 1435 2933 27 32.1 30.5 [26, 41] Surface area 1497 1433 2930 27 32.1 30.5 Subcortical volume 1495 1472 2967 25 32.0 30.6 SCZ Cortical thickness 4474 5098 9572 39 32.3a 34.5a [27, 34] Surface area 4434 5073 9507 39 32.3a 34.5a Subcortical volume 2028 2540 4568 15 34.0a 31.0a aWeighted mean not provided in paper; computed from descriptive statistics. [2, 28] aWeighted mean not provided in paper; computed from descriptive statistics. affected. As indicated by the blueness of the cells, the most prominent reductions were seen for SCZ (mean Cohen’s d across all regions = −0.22, SE = 0.014), epilepsy (mean Cohen’s d = −0.12, SE = 0.017) and BD (mean Cohen’s d = −0.097, SE = 0.011). The smallest changes were observed for MDD (mean Cohen’s d = −0.018, SE = 0.006). All regions except for the caudate and putamen exhibited signiﬁcant differences in the magnitude of Cohen’s d across disorders (Cochran’s Q p values = 0.012–2.8 × 10−32). Case–control differences in subcortical volume and cortical surface area and thickness within neuropsychiatric disorders Figure 1 presents an anatomical graph of the standardized effect sizes (Cohen’s d) measuring alterations in subcortical volume, cortical surface area and cortical thickness for 41 brain regions within seven neuropsychiatric disorders— ADHD, ASD, OCD, epilepsy, MDD, BD, and SCZ. These have been reported on prior publications. The variation in color from blue to red illustrates the phenomenon of SBRV, with some regions showing signiﬁcant reductions (blue) in volume/thickness/surface areas and others not being 2104 N. V. Radonjić et al. Fig. 1 A brain imaging plot showing standardized mean differ- ences (Cohen’s d) measuring case–control differences in sub- cortical volumes and cortical thickness for seven neuropsychiatric disorders. Results were obtained from ENIGMA working group publications. Negative values for Cohen’s d indicate smaller sizes of brain regions in cases versus unaffected comparisons. Note: ADHD attention-deﬁcit/hyperactivity disorder, ASD autism spectrum dis- order, BD bipolar disorder, MDD major depressive disorder, OCD obsessive compulsive disorder, SCZ schizophrenia. Fig. 1 A brain imaging plot showing standardized mean differ- ences (Cohen’s d) measuring case–control differences in sub- cortical volumes and cortical thickness for seven neuropsychiatric disorders. Results were obtained from ENIGMA working group publications. Negative values for Cohen’s d indicate smaller sizes of brain regions in cases versus unaffected comparisons. Note: ADHD attention-deﬁcit/hyperactivity disorder, ASD autism spectrum dis- order, BD bipolar disorder, MDD major depressive disorder, OCD obsessive compulsive disorder, SCZ schizophrenia. Fig. 1 A brain imaging plot showing standardized mean differ- ences (Cohen’s d) measuring case–control differences in sub- cortical volumes and cortical thickness for seven neuropsychiatric disorders. Results were obtained from ENIGMA working group publications. Negative values for Cohen’s d indicate smaller sizes of brain regions in cases versus unaffected comparisons. Note: ADHD attention-deﬁcit/hyperactivity disorder, ASD autism spectrum dis- order, BD bipolar disorder, MDD major depressive disorder, OCD obsessive compulsive disorder, SCZ schizophrenia. Correlation of shared genetic heritability with brain structural correlation middle frontal gyrus, individuals with ADHD showed no difference, and all other disorders showed a thinner cortex in this region compared to controls. Figure 3 shows the pairwise correlations of sMRI pheno- types and genetic overlap across each pair of neu- ropsychiatric disorders. The LD-score cross-disorder genetic correlations are positively correlated with the sMRI phenotype cross-disorder correlations (Spearman’s ρ = 0.44, p = 0.049). Leave-one-out sensitivity analyses con- ﬁrmed that the direction of the correlation was positive and remained moderate in magnitude despite removal of indi- vidual pairs of disorders from the correlation test (range of Spearman’s ρ = 0.35–0.58), except for removing SCZ/BD (Spearman’s ρ = 0.35). SCZ and BD showed the highest degree of concordance with respect to genetic and sMRI phenotype correlations. sMRI phenotype correlations among neuropsychiatric disorders For each pair of disorders, we computed the Pearson corre- lation between their sMRI phenotypes listed in Fig. 1. These are listed in Table 2 (and visualized in Fig. 2), sorted by the magnitude of the correlation. The p values reported in Table 2 are potentially downwardly biased due to inability to properly adjust for spatial coherence of nearby brain regions. Tradi- tional permutation p values are provided as a column in Table 2, which attempts to correct for potential biases due to spatial coherence. However, we were restricted from using a spatial permutation framework to generate a null distribution of correlations, because we are jointly analyzing two cortical maps (cortical thickness and surface area) that are fully overlapped. The highest positive correlation was between SCZ and BD (r = 0.81, df = 73, p < 1.3 × 10−18, Bonferroni p = 2.38 × 10−17). There were a few additional nominally signiﬁcant negative correlations, which did not survive mul- tiple testing correction: MDD and epilepsy (r = −0.37, p = 0.02), MDD and ADHD (r = −0.33, p = 0.004), SCZ and ADHD (r = −0.32, p = 0.005), ADHD and epilepsy (r = −0.36, p = 0.02), and a positive correlation between MDD and ASD (r = 0.26, p = 0.02). Discussion Disorder 1 Disorder 2 sMRI correlation Pearson’s r df se p value Boferroni adjusted p value Permutation p value BD SCZ 0.81 73 0.068 1.13E−18 2.38E−17 <1E10−4 BD MDD 0.69 73 0.085 1.21E−11 2.55E−10 <1E10−4 OCD SCZ 0.65 72 0.09 5.53E−10 1.16E−08 <1E10−4 MDD SCZ 0.58 73 0.095 5.55E−08 1.17E−06 <1E10−4 ADHD BD −0.53 73 0.099 1.18E−06 2.48E−05 <1E10−4 BD OCD 0.5 72 0.102 4.74E−06 9.95E−05 <1E10−4 MDD OCD 0.46 72 0.104 3.28E−05 6.89E−04 <1E10−4 ASD BD 0.38 73 0.108 8.98E−04 0.02 <1E10−4 ASD SCZ 0.36 73 0.109 1.35E−03 0.03 0.0176 ADHD MDD −0.33 73 0.111 4.27E−03 0.09 0.019 ADHD SCZ −0.32 73 0.111 4.63E−03 0.1 0.0014 Epilepsy MDD −0.37 39 0.149 0.02 0.38 0.0056 ADHD Epilepsy −0.36 39 0.149 0.02 0.41 0.004 ASD MDD 0.26 73 0.113 0.02 0.46 0.024 Epilepsy OCD −0.19 39 0.157 0.23 1 0.22 BD Epilepsy 0.17 39 0.158 0.3 1 0.3 ADHD OCD −0.1 72 0.117 0.39 1 0.39 ADHD ASD −0.06 73 0.117 0.6 1 0.6 Epilepsy SCZ −0.03 39 0.16 0.86 1 0.85 ASD Epilepsy 0.02 39 0.16 0.91 1 0.91 ASD OCD 0 72 0.118 0.97 1 0.97 Structural brain imaging studies offer clues about the effects of the shared genetic etiology among. . . 2105 Fig. 2 A heatmap of the cross-disorder pairwise sMRI correlations between seven neuropsychiatric disorders examined in this study. Colors in the plot correspond to the magnitude of the Pearson’s r coefﬁcients, which are provided in each tile. Note: ADHD attention- deﬁcit/hyperactivity disorder, ASD autism spectrum disorder, BD bipolar disorder, MDD major depressive disorder, OCD obsessive compulsive disorder, SCZ schizophrenia. Fig. 2 A heatmap of the cross-disorder pairwise sMRI correlations between seven neuropsychiatric disorders examined in this study. Colors in the plot correspond to the magnitude of the Pearson’s r Fig. 3 Scatter plot showing the correlation of correlations. Genetic correlations (rg) computed by LD-score regression are on the hor- izontal axis (with standard error bars), with correlations of Cohen’s d values displayed on the vertical axis. Each dot is color-coded according to the pairwise disorder correlations that were computed. Fig. 3 Scatter plot showing the correlation of correlations. Genetic correlations (rg) computed by LD-score regression are on the hor- izontal axis (with standard error bars), with correlations of Cohen’s d values displayed on the vertical axis. Each dot is color-coded according to the pairwise disorder correlations that were computed. Discussion Disorder 1 Disorder 2 sMRI correlation Pearson’s r df se p value Boferroni adjusted p value Permutation p value BD SCZ 0.81 73 0.068 1.13E−18 2.38E−17 <1E10−4 BD MDD 0.69 73 0.085 1.21E−11 2.55E−10 <1E10−4 OCD SCZ 0.65 72 0.09 5.53E−10 1.16E−08 <1E10−4 MDD SCZ 0.58 73 0.095 5.55E−08 1.17E−06 <1E10−4 ADHD BD −0.53 73 0.099 1.18E−06 2.48E−05 <1E10−4 BD OCD 0.5 72 0.102 4.74E−06 9.95E−05 <1E10−4 MDD OCD 0.46 72 0.104 3.28E−05 6.89E−04 <1E10−4 ASD BD 0.38 73 0.108 8.98E−04 0.02 <1E10−4 ASD SCZ 0.36 73 0.109 1.35E−03 0.03 0.0176 ADHD MDD −0.33 73 0.111 4.27E−03 0.09 0.019 ADHD SCZ −0.32 73 0.111 4.63E−03 0.1 0.0014 Epilepsy MDD −0.37 39 0.149 0.02 0.38 0.0056 ADHD Epilepsy −0.36 39 0.149 0.02 0.41 0.004 ASD MDD 0.26 73 0.113 0.02 0.46 0.024 Epilepsy OCD −0.19 39 0.157 0.23 1 0.22 BD Epilepsy 0.17 39 0.158 0.3 1 0.3 ADHD OCD −0.1 72 0.117 0.39 1 0.39 ADHD ASD −0.06 73 0.117 0.6 1 0.6 Epilepsy SCZ −0.03 39 0.16 0.86 1 0.85 ASD Epilepsy 0.02 39 0.16 0.91 1 0.91 ASD OCD 0 72 0.118 0.97 1 0.97 Structural brain imaging studies offer clues about the effects of the shared genetic etiology among. . . 2105 tructural brain imaging studies offer clues about the effects of the shared genetic etiology among. . . 2105 Structural brain imaging studies offer clues about the effects of the shared genetic etiology among. Table 2 Cross-disorder structural MRI phenotype correlations (ordered from smallest to largest p value) based on Cohen’s d values obtained from the ENIGMA Project. Discussion Our analysis of summary statistics from the ENIGMA ADHD, ASD, BD, MDD, OCD, SCZ, and epilepsy Working Groups and the predominantly PGC case–control GWAS identiﬁed two novel ﬁndings. First, we found substantial correlations for some disorders in the pattern of sMRI case–control differences across sub- cortical and cortical regions in line with recently pub- lished study of [23]. Second, these cross-disorder correlations in SBRV could partly be explained by the genetic correlations reported for these disorders from GWAS [3]. The cross-disorder correlations in SBRV are intriguing because, like cross-disorder genetic correlations, they sug- gest that these disorders, to varying degrees, share aspects Fig. 2 A heatmap of the cross-disorder pairwise sMRI correlations between seven neuropsychiatric disorders examined in this study. Colors in the plot correspond to the magnitude of the Pearson’s r coefﬁcients, which are provided in each tile. Note: ADHD attention- deﬁcit/hyperactivity disorder, ASD autism spectrum disorder, BD bipolar disorder, MDD major depressive disorder, OCD obsessive compulsive disorder, SCZ schizophrenia. Fig. 3 Scatter plot showing the correlation of correlations. Genetic correlations (rg) computed by LD-score regression are on the hor- izontal axis (with standard error bars), with correlations of Cohen’s d values displayed on the vertical axis. Each dot is color-coded according to the pairwise disorder correlations that were computed. The dotted line represents the best-ﬁt regression line. The Spearman’s rho (ρ) and p value are provided at the top-left corner of the panel. Note: ADHD attention-deﬁcit/hyperactivity disorder, ASD autism spectrum disorder, BD bipolar disorder, MDD major depressive dis- order, OCD obsessive compulsive disorder, SCZ schizophrenia. Table 2 Cross-disorder structural MRI phenotype correlations (ordered from smallest to largest p value) based on Cohen’s d values obtained from the ENIGMA Project. Discussion Consistent with this ﬁnding, interindividual dif- ferences in neuroimaging account for only a small amount of the variance in symptom expression or behavioral mea- sures of symptomatic or behavioral variance [24]. In the lower left region of Fig. 3, we see disorders with low genetic and low sMRI correlations. These involve correlations of epilepsy, and correlations of ADHD with all disorders except ASDs and MDD, although the latter is somewhat of an outlier. ASDs tend to have both modest genetic correlations and modest sMRI correlations with most other disorders and, hence, populates the middle range of the ﬁgure. Like the sMRI correlations among disorders, all genetic correlations with epilepsy are low, which is consistent with the low genetic correlation between neuro- logical and psychiatric disorders as reported by [2]. g p y p y The ﬁnding that SBRV correlations are correlated with genetic correlations suggests that future studies of SBRV should consider genetic sources of etiology. Yet, because only about 24% of the variance in the SBRV correlations can be accounted for by the genetic correlations, environmental sources of etiology and disease-speciﬁc genetic contributions must also be considered. These include shared confounders, such as chronicity and medication exposure, along with shared etiologic events such as birth complications or expo- sure to toxins in utero. Our prior studies of SBRV in ADHD implicated the regulation of genes in apoptosis, autophagy and neurodevelopment pathways in ADHD [42, 43]. Neu- rodevelopmental pathways had also been implicated in the cross-disorder analysis of the PCG [3], which suggests that cross-disorder similarities in these pathways may account for cross-disorder similarities in SBRV. y p The most prominent case–control differences in cortical thickness/surface area and subcortical volumes were observed for SCZ [20, 21] and BD [13, 14]. These disorders also had the highest sMRI phenotype correlations and both also showed strong sMRI phenotype correlations with MDD [16, 17] and OCD [18, 19]. As Fig. 2 shows, these disorders clustered together in the three-dimensional con- ﬁguration required to capture cross-disorder sMRI pheno- type similarity. The high sMRI correlation between SCZ and BD is consistent with prior reports of sMRI similarities between the two disorders [25]. Moreover, a large body of literature reports substantial etiologic overlap between the two disorders [26–30]. Because of such data, the SCZ and BD have been described as sharing a continuum of etiology leading to psychotic [31], neurophysiological [31] and neurocognitive [32] symptoms. Discussion MDD also has a high genetic correlation with ADHD but a negative sMRI correlation, which makes that pair an outlier in Fig. 3. high sMRI correlations. These are SCZ/BD, SZ/MDD, BD/ MDD, OCD/BD, and OCD/MDD. The inclusion of MDD in this group is notable given that it is part of the bipolar diagnosis and often occurs comorbid with other disorders. MDD also has a high genetic correlation with ADHD but a negative sMRI correlation, which makes that pair an outlier in Fig. 3. of their etiology and pathophysiology. Any interpretation of the cross-disorder sMRI correlations must keep in mind that, for all disorders, the case–control differences in sMRI measures are small (Fig. 1). The largest Cohen’s d values are only −0.5 for SCZ [20, 21], −0.4 for epilepsy [15], −0.3 for BD [13, 14], −0.2 for ADHD [10, 11] and ASDs [12], and −0.1 for MDD [16, 17] and OCD [18, 19]. These small case–control differences are consistent with results from GWAS and environmental risk studies, which speaks to the fact that the effects of common risk factors are, with some rare exceptions, individually small. Although it is conceivable that these small risks could accumulate to create a more dramatic pathophysiology in the brain, the ENIGMA data show that this is not the case for sMRI measures. Consistent with this ﬁnding, interindividual dif- ferences in neuroimaging account for only a small amount of the variance in symptom expression or behavioral mea- sures of symptomatic or behavioral variance [24]. of their etiology and pathophysiology. Any interpretation of the cross-disorder sMRI correlations must keep in mind that, for all disorders, the case–control differences in sMRI measures are small (Fig. 1). The largest Cohen’s d values are only −0.5 for SCZ [20, 21], −0.4 for epilepsy [15], −0.3 for BD [13, 14], −0.2 for ADHD [10, 11] and ASDs [12], and −0.1 for MDD [16, 17] and OCD [18, 19]. These small case–control differences are consistent with results from GWAS and environmental risk studies, which speaks to the fact that the effects of common risk factors are, with some rare exceptions, individually small. Although it is conceivable that these small risks could accumulate to create a more dramatic pathophysiology in the brain, the ENIGMA data show that this is not the case for sMRI measures. Discussion The dotted line represents the best-ﬁt regression line. The Spearman’s rho (ρ) and p value are provided at the top-left corner of the panel. Note: ADHD attention-deﬁcit/hyperactivity disorder, ASD autism spectrum disorder, BD bipolar disorder, MDD major depressive dis- order, OCD obsessive compulsive disorder, SCZ schizophrenia. Fig. 3 Scatter plot showing the correlation of correlations. Genetic correlations (rg) computed by LD-score regression are on the hor- izontal axis (with standard error bars), with correlations of Cohen’s d values displayed on the vertical axis. Each dot is color-coded according to the pairwise disorder correlations that were computed. The dotted line represents the best-ﬁt regression line. The Spearman’s rho (ρ) and p value are provided at the top-left corner of the panel. Note: ADHD attention-deﬁcit/hyperactivity disorder, ASD autism spectrum disorder, BD bipolar disorder, MDD major depressive dis- order, OCD obsessive compulsive disorder, SCZ schizophrenia. Fig. 3 Scatter plot showing the correlation of correlations. Genetic Fig. 3 Scatter plot showing the correlation of correlations. Genetic correlations (rg) computed by LD-score regression are on the hor- izontal axis (with standard error bars), with correlations of Cohen’s d values displayed on the vertical axis. Each dot is color-coded according to the pairwise disorder correlations that were computed. The dotted line represents the best-ﬁt regression line. The Spearman’s rho (ρ) and p value are provided at the top-left corner of the panel. Note: ADHD attention-deﬁcit/hyperactivity disorder, ASD autism spectrum disorder, BD bipolar disorder, MDD major depressive dis- order, OCD obsessive compulsive disorder, SCZ schizophrenia. Fig. 2 A heatmap of the cross-disorder pairwise sMRI correlations between seven neuropsychiatric disorders examined in this study. Colors in the plot correspond to the magnitude of the Pearson’s r coefﬁcients, which are provided in each tile. Note: ADHD attention- deﬁcit/hyperactivity disorder, ASD autism spectrum disorder, BD bipolar disorder, MDD major depressive disorder, OCD obsessive compulsive disorder, SCZ schizophrenia. correlations in SBRV could partly be explained by the genetic correlations reported for these disorders from GWAS [3]. The cross-disorder correlations in SBRV are intriguing because, like cross-disorder genetic correlations, they sug- gest that these disorders, to varying degrees, share aspects N. V. Radonjić et al. 2106 high sMRI correlations. These are SCZ/BD, SZ/MDD, BD/ MDD, OCD/BD, and OCD/MDD. The inclusion of MDD in this group is notable given that it is part of the bipolar diagnosis and often occurs comorbid with other disorders. Discussion The ENPACT study [33] showed shared fronto–temporo–occipital gray matter volume deﬁcits in the right hemisphere of two disorders. A systematic review of associations between functional MRI activity and polygenic risk for SCZ and BD [26] reported that genetic load for these disorders affects task-related recruitment of predominantly frontal lobe brain regions. Although we used data derived from very large samples (ENIGMA, iPSYCH, and the PGC), several limitations moderate the strength of our conclusions. We inherit all the limitations of the constituent studies, but are further limited because we analyzed summary statistics, not the original data, which would require the sharing of individual subject level data, an ongoing effort among the ENIGMA disorder working groups. Thus, we cannot determine whether the possible use of controls shared among studies affected our results. It is also possible that some research participants were included in the genetic and sMRI data sets for the same disorder. The p value obtained by our Spearman’s correlation test of cross-disorder sMRI and genetic corre- lations may be inaccurate due to spatial autocorrelation among sMRI Cohen’s d estimates, which can downwardly bias standard errors and lead to deﬂated p values. Con- sidering we are not able to completely address with auto- correlation among brain regions using summary statistics alone, the p value from our primary analysis (presented in Many studies have reported that OCD can be a comorbid diagnosis with SCZ or that patients with SCZ can have OCD symptoms [34–41]. Presented ﬁndings of a signiﬁcant over- lap in sMRI phenotypes along with the known SCZ/OCD genetic correlations suggests that more work should examine shared pathophysiologic features between these disorders and should assess the degree to which confounds, such as med- ication status or chronicity, might explain these results. The sMRI phenotype correlations mirror, to some extent, the cross-disorder correlations from GWAS. Figure 3 shows a modest, yet distinct, linear correlation between the sMRI phenotype and genetic correlations. In the upper right-hand section of the plot, we see disorders having high genetic and Structural brain imaging studies offer clues about the effects of the shared genetic etiology among. . . 2107 Fig. 3) should be interpreted with caution. Another problem is that we could not address effects of medications or chronicity on brain structure. Furthermore, for some of the disorders, we could use youth and adult data, whereas for others only adult effect data were used. Discussion Because ﬁndings can differ substantially depending on the age range of the samples included (e.g., [10, 11, 18, 19], this might have inﬂuenced our ﬁndings. For these reasons, analyses of participant level data will be needed to address these issues to draw stronger and more detailed conclusions. We also did not have any longitudinal data available, which limits the ability to test hypotheses about shared and unique developmental trajectories among disorders. Acknowledgements SVF is supported by the European Union’s Seventh Framework Program for research, technological development and demonstration under grant agreement no. 602805, the European Union’s Horizon 2020 research and innovation program under grant agreements nos. 667302 and 728018 and NIMH grants 5R01MH101519 and U01 MH109536-01. Research Council of Nor- way (#223273). BF is supported by a personal Vici grant from the Netherlands Organization for Scientiﬁc Research (NWO, grant number 91813669) and by a grant from the European Union’s Horizon 2020 program for the CoCa project (grant agreement no 667302). ENIGMA work is supported by NIH grants U54 EB020403 (PI: PT), R01 MH116147 (PI: PT) and R01MH117601 (MPIs: NJ and LS). MH is supported by a personal Veni grant from the Netherlands Organization for Scientiﬁc Research (NWO, grant number 91619115). CMD is supported by NIH grants R01 NS065838 and R21 NS107739. PR is a recipient of a pre-doctoral fellowship from the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR), Generalitat de Catalu- nya, Spain (2016FI_B 00899). LS is supported by a NHMRC Career Development Fellowship (1140764). SMS is supported by Epilepsy Society, UK, and the work was partly undertaken at UCLH/UCL, which received a proportion of funding from the UK Department of Health’s NIHR Biomedical Research Centers funding scheme.). TGMVE is supported by NIH grants U54 EB020403 (PI: PT), R01 MH116147 (PI: PT), R01MH117601 (MPIs: NJ and LS), and R01MH121246 (MPIs: Turner, TGMVE, and Calhoun). Despite these limitations, we have documented cross- disorder correlations in SBRV as assessed by sMRI. These cross-disorder SBRV correlations are positively associated with the disorders’ corresponding cross-disorder genetic correlations. This ﬁnding is a novel contribution worthy of further study that contributes to novel body of literature focused on cross-level correspondence of genetic and neu- roimaging presentations of different psychiatric disorders [44–49]. Our work supports conclusions from previous GWAS studies suggesting a partially shared etiology and pathophysiology among many disorders [2, 50]. Compliance with ethical standards Conﬂict of interest OA has received speaker’s honorarium from Lundbeck and is a consultant to HealthLytix. In the past year, SVF received income, potential income, travel expenses continuing edu- cation support, and/or research support from Tris, Otsuka, Arbor, Ironshore, Shire, Akili Interactive Labs, Enzymotec, Sunovion, Supernus and Genomind. With his institution, he has US patent US20130217707 A1 for the use of sodium–hydrogen exchange inhi- bitors in the treatment of ADHD. He also receives royalties from books published by Guilford Press: Straight Talk about Your Child’s Mental Health, Oxford University Press: Schizophrenia: The Facts and Elsevier: ADHD: Non-Pharmacologic Interventions. He is Program Director of www.adhdinadults.com. BF received educational speaking fees from Medice. All other authors declare no conﬂict of interest. Data availability URLs for GWAS SCZ from ckqny.scz2snpres.gz (https://www.med.unc.edu/pgc/results-and-downloads), ASD from iPSYCH-PGC_ASD_Nov2017.gz (https://www.med. unc.edu/pgc/results-and-downloads), OCD from PGC_OC- D_Aug2017-20171122T182645Z-001.zip > ocd_aug2017.gz (https://www.med.unc.edu/pgc/results-and-downloads), ADHD from adhd_ul2017.gz (https://www.med.unc.edu/pgc/ results-and-downloads), BD from daner_PGC_BIP32b_md- s7a_0416a.gz (https://www.med.unc.edu/pgc/results-and-dow nloads), Epilepsy from all_epilepsy_METAL.gz (http://www. epigad.org/gwas_ilae2018_16loci.html), and MDD from PGC_UKB_depression_genome-wide.txt (https://doi.org/10. 7488/ds/2458). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 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Faraone 2 Department of Psychiatry, SUNY Upstate Medical University, Syracuse, NY, USA Departments of Psychiatry and of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY, USA Psychiatric Genetics Unit, Group of Psychiatry, Mental Health and Addiction, Vall d’Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain Department of Psychiatry, Hospital Universitari Vall d’Hebron, Barcelona, Spain 5 NORMENT—Institute of Clinical Medicine, Division of Mental Health and Addiction, Oslo University Hospital, University of Oslo, Oslo, Norway 6 Radboudumc, Radboud University Medical Center, Nijmegen, The Netherlands 7 Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands 8 Department of Cognitive Neuroscience, Radboud University Medical Center, Nijmegen, The Netherlands Nevena V. Radonjić 1 ●Jonathan L. Hess 2 ●Paula Rovira 3,4 ●Ole Andreassen5 ●Jan K. Buitelaar 6,7,8 ● Christopher R. K. Ching9 ●Barbara Franke 7,10,11 ●Martine Hoogman10 ●Neda Jahanshad 12 ●Carrie McDonald13 ● Lianne Schmaal 14,15 ●Sanjay M. Sisodiya16,17 ●Dan J. Stein 18 ●Odile A. van den Heuvel19 ● Theo G. M. van Erp 20,21 ●Daan van Rooij22 ●Dick J. Veltman19 ●Paul Thompson23 ●Stephen V. Faraone 2 Nevena V. Radonjić 1 ●Jonathan L. Hess 2 ●Paula Rovira 3,4 ●Ole Andreassen5 ●Jan K. Buitelaar 6,7,8 ● Christopher R. K. Ching9 ●Barbara Franke 7,10,11 ●Martine Hoogman10 ●Neda Jahanshad 12 ●Carrie McDonald13 ● Lianne Schmaal 14,15 ●Sanjay M. Sisodiya16,17 ●Dan J. Stein 18 ●Odile A. van den Heuvel19 ● Theo G. M. van Erp 20,21 ●Daan van Rooij22 ●Dick J. Veltman19 ●Paul Thompson23 ●Stephen V. 4 Department of Psychiatry, Hospital Universitari Vall d’Hebron, Barcelona, Spain Afﬁliations Faraone 2 1 Department of Psychiatry, SUNY Upstate Medical University, Syracuse, NY, USA 5 NORMENT—Institute of Clinical Medicine, Division of Mental Health and Addiction, Oslo University Hospital, University of Oslo, Oslo, Norway 2 Departments of Psychiatry and of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY, USA 6 Radboudumc, Radboud University Medical Center, Nijmegen, The Netherlands 3 Psychiatric Genetics Unit, Group of Psychiatry, Mental Health and Addiction, Vall d’Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain 7 Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands 4 Department of Psychiatry, Hospital Universitari Vall d’Hebron, Barcelona, Spain 8 Department of Cognitive Neuroscience, Radboud University Medical Center, Nijmegen, The Netherlands 8 Department of Cognitive Neuroscience, Radboud University Medical Center, Nijmegen, The Netherlands 4 Department of Psychiatry, Hospital Universitari Vall d’Hebron, Barcelona, Spain 2110 N. V. Radonjić et al. 23 Neuro Imaging Institute for Neuroimaging and Informatics, Keck School of Medicine of the University of Southern California, Marina Del Rey, CA, USA Afﬁliations 9 Imaging Genetics Center, USC Mark and Mary Stevens Neuroimaging and Informatics Institute, Keck School of Medicine of the University of Southern California, Marina Del Rey, CA, USA 10 Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands 11 Department of Psychiatry, Radboud University Medical Center, Nijmegen, The Netherlands 12 Imaging Genetics Center, Department of Neurology and Biomedical Engineering, USC Mark and Mary Stevens Neuroimaging and Informatics Institute, Keck School of Medicine of USC, University of Southern California, Marina Del Rey, CA, USA 13 Department of Psychiatry, Center for Multimodal Imaging and Genetics (CMIG), University of California, San Diego, CA, USA 14 Centre for Youth Mental Health, The University of Melbourne, Parkville, VIC, Australia 15 Orygen, The National Centre of Excellence for Youth Mental Health, Parkville, VIC, Australia 16 UCL Queen Square Institute of Neurology, Department of Clinical and Experimental Epilepsy, University College London, London, UK 17 Chalfont Centre for Epilepsy, Epilepsy Society, Bucks, UK 18 SA MRC Unit on Risk & Resilience in Mental Disorders, Department of Psychiatry & Neuroscience Institute, University of Cape Town, Cape Town, South Africa 19 Department of Psychiatry and Department of Anatomy & Neurosciences, Amsterdam UMC/VUmc, Amsterdam, The Netherlands 20 Clinical Translational Neuroscience Laboratory, Department of Psychiatry and Human Behavior, University of California Irvine, Irvine, CA, USA 21 Center for the Neurobiology of Learning and Memory, University of California Irvine, Irvine, CA, USA 22 Donders Centre for Cognitive Neuroimaging, Radboud University Medical Center, Nijmegen, The Netherlands 23 Neuro Imaging Institute for Neuroimaging and Informatics, Keck School of Medicine of the University of Southern California, Marina Del Rey, CA, USA 16 UCL Queen Square Institute of Neurology, Department of Clinical and Experimental Epilepsy, University College London, London, UK 17 Chalfont Centre for Epilepsy, Epilepsy Society, Bucks, UK 18 SA MRC Unit on Risk & Resilience in Mental Disorders, Department of Psychiatry & Neuroscience Institute, University of Cape Town, Cape Town, South Africa 19 Department of Psychiatry and Department of Anatomy & Neurosciences, Amsterdam UMC/VUmc, Amsterdam, The Netherlands 20 Clinical Translational Neuroscience Laboratory, Department of Psychiatry and Human Behavior, University of California Irvine, Irvine, CA, USA 21 Center for the Neurobiology of Learning and Memory, University of California Irvine, Irvine, CA, USA 22 Donders Centre for Cognitive Neuroimaging, Radboud University Medical Center, Nijmegen, The Netherlands 23 Neuro Imaging Institute for Neuroimaging and Informatics, Keck School of Medicine of the University of Southern California, Marina Del Rey, CA, USA 9 Imaging Genetics Center, USC Mark and Mary Stevens Neuroimaging and Informatics Institute, Keck School of Medicine of the University of Southern California, Marina Del Rey, CA, USA 10 Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands 11 Department of Psychiatry, Radboud University Medical Center, Nijmegen, The Netherlands 12 Imaging Genetics Center, Department of Neurology and Biomedical Engineering, USC Mark and Mary Stevens Neuroimaging and Informatics Institute, Keck School of Medicine of USC, University of Southern California, Marina Del Rey, CA, USA 13 Department of Psychiatry, Center for Multimodal Imaging and Genetics (CMIG), University of California, San Diego, CA, USA 14 Centre for Youth Mental Health, The University of Melbourne, Parkville, VIC, Australia 15 Orygen, The National Centre of Excellence for Youth Mental Health, Parkville, VIC, Australia 16 UCL Queen Square Institute of Neurology, Department of Clinical and Experimental Epilepsy, University College London, London, UK 9 Imaging Genetics Center, USC Mark and Mary Stevens Neuroimaging and Informatics Institute, Keck School of Medicine of the University of Southern California, Marina Del Rey, CA, USA 17 Chalfont Centre for Epilepsy, Epilepsy Society, Bucks, UK 17 Chalfont Centre for Epilepsy, Epilepsy Society, Bucks, UK 10 Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands 18 SA MRC Unit on Risk & Resilience in Mental Disorders, Department of Psychiatry & Neuroscience Institute, University of Cape Town, Cape Town, South Africa 11 Department of Psychiatry, Radboud University Medical Center, Nijmegen, The Netherlands 19 Department of Psychiatry and Department of Anatomy & Neurosciences, Amsterdam UMC/VUmc, Amsterdam, The Netherlands 12 Imaging Genetics Center, Department of Neurology and Biomedical Engineering, USC Mark and Mary Stevens Neuroimaging and Informatics Institute, Keck School of Medicine of USC, University of Southern California, Marina Del Rey, CA, USA 20 Clinical Translational Neuroscience Laboratory, Department of Psychiatry and Human Behavior, University of California Irvine, Irvine, CA, USA 20 Clinical Translational Neuroscience Laboratory, Department of Psychiatry and Human Behavior, University of California Irvine, Irvine, CA, USA 13 Department of Psychiatry, Center for Multimodal Imaging and Genetics (CMIG), University of California, San Diego, CA, USA 21 Center for the Neurobiology of Learning and Memory, University of California Irvine, Irvine, CA, USA 21 Center for the Neurobiology of Learning and Memory, University of California Irvine, Irvine, CA, USA 14 Centre for Youth Mental Health, The University of Melbourne, Parkville, VIC, Australia 22 Donders Centre for Cognitive Neuroimaging, Radboud University Medical Center, Nijmegen, The Netherlands 23 Neuro Imaging Institute for Neuroimaging and Informatics, Keck School of Medicine of the University of Southern California, Marina Del Rey, CA, USA
https://openalex.org/W2883718081	https://openaccess.city.ac.uk/id/eprint/20346/3/Hobby2018_Article_EffectOfVaryingSkinSurfaceElec.pdf	English	null	Effect of varying skin surface electrode position on electroretinogram responses recorded using a handheld stimulating and recording system	Documenta ophthalmologica	2,018	cc-by	4,814	City Research Online City Research Online City, University of London Institutional Repository Citation: Hobby, A. E., Kozareva, D., Yonova-Doing, E., Hossain, I. T., Katta, M., Huntjens, B., Hammond, C. J., Binns, A. M. & Mahroo, O. A. (2018). Effect of varying skin surface electrode position on electroretinogram responses recorded using a handheld stimulating and recording system. Documenta Ophthalmologica, 137(2), pp. 79-86. doi: 10.1007/s10633-018-9652-z This is the published version of the paper. This version of the publication may differ from the final published version Permanent repository link: https://openaccess.city.ac.uk/id/eprint/20346/ Link to published version: https://doi.org/10.1007/s10633-018-9652-z Copyright: City Research Online aims to make research outputs of City, University of London available to a wider audience. Copyright and Moral Rights remain with the author(s) and/or copyright holders. URLs from City Research Online may be freely distributed and linked to. Reuse: Copies of full items can be used for personal research or study, educational, or not-for-profit purposes without prior permission or charge. Provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way. City Research Online: http://openaccess.city.ac.uk/ publications@city.ac.uk City Research Online: http://openaccess.city.ac.uk/ publications@city.ac.uk City Research Online: Doc Ophthalmol https://doi.org/10.1007/s10633-018-9652-z (0123456789().,-volV)(0123456789().,-volV) ORIGINAL RESEARCH ARTICLE Effect of varying skin surface electrode position on electroretinogram responses recorded using a handheld stimulating and recording system Angharad E. Hobby . Diana Kozareva . Ekaterina Yonova-Doing . Ibtesham T. Hossain . Mohamed Katta . Byki Huntjens . Christopher J. Hammond . Alison M. Binns . Omar A. Mahroo Received: 12 February 2018 / Accepted: 17 July 2018 The Author(s) 2018 Received: 12 February 2018 / Accepted: 17 July 2018 The Author(s) 2018 Light-adapted ﬂicker and ﬂash ERGs were recorded for an additional 10 healthy subjects in two consec- utive recording sessions: in the test eye, electrode position was varied from 2 to 10–20 mm below the lid margin between sessions; in the fellow (control) eye, the electrode was 2 mm below the lid margin throughout. Amplitudes and peak times (test eye normalised to control eye) were compared for the two sessions. A. E. Hobby D. Kozareva E. Yonova-Doing C. J. Hammond O. A. Mahroo Department of Twin Research and Genetic Epidemiology, King’s College London, St Thomas’ Hospital Campus, London SE1 7EH, UK A. E. Hobby D. Kozareva E. Yonova-Doing I. T. Hossain M. Katta C. J. Hammond O. A. Mahroo Department of Ophthalmology, King’s College London, St Thomas’ Hospital Campus, London SE1 7EH, UK Abstract Purpose A handheld device (the RETeval system, LKC Technologies) aims to increase the ease of electroretinogram (ERG) recording by using specially designed skin electrodes, rather than corneal elec- trodes. We explored effects of electrode position on response parameters recorded using this device. Methods Healthy adult twins were recruited from the TwinsUK cohort and underwent recording of light- adapted ﬂicker ERGs (corresponding to international standard stimuli). In Group 1, skin electrodes were placed in a ‘‘comfortable’’ position, which was up to 20 mm below the lid margin. For subsequent partic- ipants (Group 2), the electrode was positioned 2 mm from the lid margin as recommended by the manu- facturer. Amplitudes and peak times (averaged from both eyes) were compared between groups after age- matching and inclusion of only one twin per pair. Results Including one twin per pair, and age-match- ing yielded 28 individuals per group. Flicker ERG amplitudes were signiﬁcantly lower for Group 1 than Group 2 participants (p = 0.0024). However, mean peak times did not differ between groups (p = 0.54). For the subjects in whom electrode position was changed between recording sessions, ﬂash and ﬂicker amplitudes were signiﬁcantly lower when positioned A. E. Hobby D. Kozareva E. Yonova-Doing I. T. Hossain M. Katta C. J. Hammond O. A. Mahroo Department of Ophthalmology, King’s College London, St Thomas’ Hospital Campus, London SE1 7EH, UK O. A. Mahroo (&) UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK e-mail: omar.mahroo@moorﬁelds.nhs.uk A. E. Hobby B. Huntjens A. M. Binns Centre of Applied Vision Research, City, University of London, Northampton Square, London EC1V 0HB, UK O. A. Mahroo Medical Retina Service, Moorﬁelds Eye Hospital, 162 City Road, London EC1V 2PD, UK A. E. Hobby D. Kozareva E. Yonova-Doing C. J. Hammond O. A. Mahroo Department of Twin Research and Genetic Epidemiology, King’s College London, St Thomas’ Hospital Campus, London SE1 7EH, UK O. A. Mahroo Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3EG, UK 12 123 Doc Ophthalmol further from the lid margin (p \ 0.005), but peak times were similar (p [ 0.5). described in 1942 [4], and reported in several publi- cations since (references [5–11] are a selection). Abstract Response amplitudes are substantially smaller than those recorded with corneal electrodes [8–11], and effects of different electrode positions, as well as directions of gaze, have been examined [5, 6, 9]: substantial changes in amplitude are observed in different recording positions. Conclusions Moving the skin electrodes further from the lid margin signiﬁcantly reduces response amplitudes, highlighting the importance of consistent electrode positioning. However, this does not signif- icantly affect peak times. Thus, it may be feasible to adopt a more comfortable position in participants who cannot tolerate the recommended position if analysis is restricted to peak time parameters. Given the special design of the RETeval skin electrodes, incorporating active, reference and ground electrodes in a single strip, and their increasing use worldwide, we sought to examine, in this speciﬁc case, the effect of the different electrode positions on amplitudes and peak times of standard light-adapted responses (designed to correspond to ISCEV (Inter- national Society for the Clinical Electrophysiology of Vision) standard stimuli [12]) recorded using the RETeval stimulator. Keywords Electroretinogram Retina Electrode Retinal function Keywords Electroretinogram Retina Electrode Retinal function Introduction The RETevalTM (LKC Technologies, Inc., Gaithers- burg, MD, USA) is a handheld device for recording the full-ﬁeld electroretinogram (ERG), which aims to improve the ease of ERG recording in a general clinical or ofﬁce environment [1–3]. Equipped with a small Ganzfeld dome, an infrared LED and camera system (for monitoring the subject’s eye) and skin electrodes, it is designed to record monocular ERGs. An inbuilt pupilometer measures pupil diameter and adjusts stimulus strength accordingly to achieve consistent retinal illuminance, allowing ERGs to be recorded with natural pupils. 123 Methods As part of a larger ongoing study, exploring genetic and environmental factors affecting ERG recordings in health and disease, adult twins were recruited from the TwinsUK cohort based at St Thomas’ Hospital London [13] and underwent recording of light-adapted ﬂicker ERGs using the handheld stimulator. Fig. 1 Two different electrode positions in a study participant. The skin sensor electrodes are placed 2 mm below the lid margin (manufacturer’s recommended position) in the upper photograph, and in an inferior position 10–20 mm below lid margin (lower photograph). Permission granted for use of photograph To minimise invasiveness, the device is designed for use with skin surface ‘‘sensor strips’’. These disposable, single use skin surface electrodes are speciﬁc to the right and left eye, respectively. The manufacturer recommends that the nasal edge of the sensor strip should be aligned with the midline of the pupil and the superior edge of the strip should be placed 2 mm below the patient’s lower lid margin (Fig. 1). This type of electrode generally requires little application time and has anecdotally proven to be more comfortable for patients undergoing ERGs than electrodes that make contact with the ocular surface. However, some subjects report mild discomfort with the recommended position due to proximity to eyelashes and interference with eye closure, and an alternative electrode position, further from the lid margin, over the orbital rim, can be more comfortable. Fig. 1 Two different electrode positions in a study participant. The skin sensor electrodes are placed 2 mm below the lid margin (manufacturer’s recommended position) in the upper photograph, and in an inferior position 10–20 mm below lid margin (lower photograph). Permission granted for use of photograph The use of periorbital skin surface electrodes to record the electroretinogram has a long history, ﬁrst 123 123 Doc Ophthalmol Participants gave informed consent; the study had ethics committee approval and adhered to the tenets of the Declaration of Helsinki. but remained in the recommended position (2 mm below the lid margin) in the fellow eye. ERG parameters from the test eye were normalised to those of the control eye (test eye divided by control eye) to control for any adaptational changes between the two recording sessions, and the resulting normalised parameters compared between sessions (Wilcoxon signed rank test). Intergroup comparison In the ﬁrst group, 48 consecutive twin participants underwent recordings. Including only one twin per pair yielded 28 participants, mean (SD) age 60.3 (10.0) years (median 59.0; minimum 43.2; maximum 76.6; interquartile range 17.1 years); all were female (re- ﬂecting the demographics of the TwinsUK cohort which is overwhelmingly female). In the second group of 200 consecutive participants, including only one twin per pair gave 122 participants, mean (SD) age 46.7 (16.8) years; 14.7% were male. The mean ages were signiﬁcantly different (p \ 0.0001). The cohorts were then age- and sex-matched by ﬁrst removing the males from the second group, and then including the same numbers of participants per decade as in the ﬁrst group (by excluding any excess participants in each decade in the second group in chronological order of recruitment). The mean age of the remaining 28 participants in the second group was now 61.0 (9.4) years (median 63.3; minimum 43.4; maximum 77.5; interquartile range 16.7 years), which was not signif- icantly different from Group 1 (p = 0.805). In the ﬁrst 48 consecutively recruited participants (Group 1), skin electrodes were placed in a ‘‘comfort- able’’ position, ranging from near the lid margin to approximately 2 cm below the lid margin. In the next 200 consecutive participants (Group 2), electrodes were positioned in the manufacturer recommended location (Fig. 1). As ERG parameters from both twins of a given pair are highly correlated [14], only one twin from each pair was included for subsequent analysis. As ERG parameters vary with age [14, 15], the two groups were age-matched by ensuring the same numbers of partic- ipants in each age decade. This resulted in 28 participants per group. ERG parameters (ﬂicker amplitudes and peak times), averaged from both eyes, were compared between the two age-matched groups (Mann–Whitney test). Table 1 compares ﬂicker amplitudes and implicit times for the two groups. Amplitudes were signiﬁ- cantly lower in Group 1 compared with Group 2 (p = 0.0024) whilst peak times did not differ signif- icantly (p = 0.54). Figure 2 compares the distribution of values between the two groups as boxplots. The median amplitude was 41% lower for Group 1 compared with Group 2. Methods Stimuli were delivered through natural pupils, and strengths were automatically adjusted by the device according to pupil diameter to correspond to interna- tional standard stimuli. The ISCEV standard speciﬁes the delivery of 3.0 photopic cd m-2 s white stimuli in the presence of a 30 cd m-2 white background [12]. The device delivers 85 Td s stimuli [chromaticity (0.33, 0.33)] on a background of 850 Td (which would correspond to the ISCEV standard strength stimulus delivered through a 6-mm-diameter pupil). The white ﬂicker and background stimuli were produced by simultaneous activation of red, green and blue LEDs. Flicker stimuli were delivered at 28.3 Hz, and the response was an average of 141–424 presentations. In most cases, each stimulus run was repeated once, and the parameters averaged. Participants were already light-adapted in standard indoor incandescent illumi- nation (approximately 40–100 photopic cd m-2). Intergroup comparison If right and left eyes were compared separately, the same pattern was seen: amplitudes were signiﬁcantly lower in Group 1 (p = 4.8 9 10-4 and 0.012 for right and left eyes, respectively), whilst peak times were not signiﬁcantly different (p = 0.90 and 0.18 for right and left eyes, respectively). In a third group of healthy participants (n = 10), photopic ﬂash (85 Td s, 2 Hz) and ﬂicker ERGs (as above) were recorded. Thirty ﬂashes were delivered per run, and the series was repeated, so ﬁnal param- eters were typically the average from 60 consecutive ﬂash presentations. The duration of each ﬂash was less than 5 ms. The following protocol was undertaken in two consecutive recording sessions: in one session, electrodes were placed in the recommended position for both eyes; in the other session, the electrode was placed 10–20 mm below the lid margin in the test eye, 12 3 3 Doc Ophthalmol [age range 22.9–45.3 years; mean (SD) 33.1 (7.1) years; median 34.2 years]. Sample recordings from one participant are shown in Fig. 3: the waveforms were similar in both recording positions, but the response was substantially larger in the lid margin position. Green and orange lines show averages of consecutive runs of stimulus presentations, showing reasonably good intra-session reproducibility. Fig- ure 4 shows distributions for amplitudes and peak times for the group (test eye normalised to fellow eye as described in ‘‘Methods’’). Again, amplitudes were signiﬁcantly lower when the electrode was 10–20 mm, compared with 2 mm, from the lid margin, but peak times were similar; p values are If, instead of age-matching the groups, parameters were compared between the two electrode positions adjusting for age as a covariant (28 participants from the ﬁrst group, and 117 from the second group in whom measurements from both eyes were available), amplitudes were again found to be lower in the ﬁrst group, but peak times were similar (p = 0.0001 and 0.36 for averaged amplitude and peak time compar- isons, respectively). Intra-subject comparison In the second part of the study, 10 healthy volunteers (4 male; 6 female) underwent consecutive recordings Table 1 Parameter values compared between the two age-matched groups Group 1 (‘‘comfortable’’ electrode position) Group 2 (recommended electrode position) p value for comparison (Mann–Whitney) Mean (SD) Median (LQ, UQ) Mean (SD) Median (LQ, UQ) Flicker ERG amplitude (microvolts) 21.0 (12.0) 18.6 (10.7, 28.7) 30.6 (9.7) 31.7 (23.3, 37.9) 0.0024* Flicker ERG peak time (ms) 25.9 (1.4) 25.5 (25.0, 26.6) 25.9 (0.9) 26.0 (25.2, 26.7) 0.54 p \ 0.05 regarded as signiﬁcant Fig. 2 Boxplots comparing the distribution of ERG parameters between participants in Groups 1 and 2. In Group 1, the electrode was in a comfortable position (up to 20 mm from the lid margin); in Group 2, the electrode was placed in the recommended position of 2 mm below the lid margin. Median, upper and lower quartiles (limits of box) are shown. Squares plot means; whiskers span the 5th–95th centiles; and crosses plot minimum and maximum data points. Amplitudes are plotted in a and peak times in b Table 1 Parameter values compared between the two age-matched groups Group 1 (‘‘comfortable’’ electrode position) Group 2 (recommended electrode position) p value for comparison (Mann–Whitney) Mean (SD) Median (LQ, UQ) Mean (SD) Median (LQ, UQ) Flicker ERG amplitude (microvolts) 21.0 (12.0) 18.6 (10.7, 28.7) 30.6 (9.7) 31.7 (23.3, 37.9) 0.0024 Flicker ERG peak time (ms) 25.9 (1.4) 25.5 (25.0, 26.6) 25.9 (0.9) 26.0 (25.2, 26.7) 0.54 p \ 0.05 regarded as signiﬁcant Table 1 Parameter values compared between the two age-matched groups Group 1 (‘‘comfortable’’ electrode position) Group 2 (recommended electrode position) p value for comparison (Mann–Whitney) Mean (SD) Median (LQ, UQ) Mean (SD) Median (LQ, UQ) Flicker ERG amplitude (microvolts) 21.0 (12.0) 18.6 (10.7, 28.7) 30.6 (9.7) 31.7 (23.3, 37.9) 0.0024 Flicker ERG peak time (ms) 25.9 (1.4) 25.5 (25.0, 26.6) 25.9 (0.9) 26.0 (25.2, 26.7) 0.54 *p \ 0.05 regarded as signiﬁcant Table 1 Parameter values compared between the two age-matched groups Fig. 2 Boxplots comparing the distribution of ERG parameters between participants in Groups 1 and 2. In Group 1, the electrode was in a comfortable position (up to 20 mm from the lid margin); in Group 2, the electrode was placed in the recommended position of 2 mm below the lid margin. Median, upper and lower quartiles (limits of box) are shown. Intra-subject comparison Squares means; whiskers span the 5th–95th centiles; and crosses minimum and maximum data points. Amplitudes are plotte a and peak times in b upper and lower quartiles (limits of box) are shown. Squares plot means; whiskers span the 5th–95th centiles; and crosses plot minimum and maximum data points. Amplitudes are plotted in a and peak times in b upper and lower quartiles (limits of box) are shown. Squares plot means; whiskers span the 5th–95th centiles; and crosses plot minimum and maximum data points. Amplitudes are plotted in a and peak times in b Fig. 2 Boxplots comparing the distribution of ERG parameters between participants in Groups 1 and 2. In Group 1, the electrode was in a comfortable position (up to 20 mm from the lid margin); in Group 2, the electrode was placed in the recommended position of 2 mm below the lid margin. Median, 12 123 Doc Ophthalmol given in the ﬁgure legend. Median amplitudes were 44–53% lower in the second position compared with the ﬁrst. reduction in response amplitudes (by approximately 40–50%) although peak times appeared to be unaf- fected. This ﬁnding emphasises the importance of consistent skin electrode positioning, particularly h lit d b i l d Fig. 3 Sample traces from one participant. a, b Averaged light- adapted ERGs to a ﬂash obtained from right and left eyes, respectively. In the right eye, the skin electrode was placed 2 mm from the lid margin. In the left eye, the electrode was 15 mm below the lid margin. c, d Light-adapted responses to the ﬂicker stimulus obtained with electrodes placed as in a, b, respectively. Green and orange traces represent averaged recordings obtained from consecutive runs of stimulus presentations ﬂicker stimulus obtained with electrodes placed as in a, b, respectively. Green and orange traces represent averaged recordings obtained from consecutive runs of stimulus presentations Fig. 3 Sample traces from one participant. a, b Averaged light- adapted ERGs to a ﬂash obtained from right and left eyes, respectively. In the right eye, the skin electrode was placed 2 mm from the lid margin. In the left eye, the electrode was 15 mm below the lid margin. c, d Light-adapted responses to the given in the ﬁgure legend. Median amplitudes were 44–53% lower in the second position compared with the ﬁrst. reduction in response amplitudes (by approximately 40–50%) although peak times appeared to be unaf- fected. Intra-subject comparison This ﬁnding emphasises the importance of consistent skin electrode positioning, particularly when amplitudes are being analysed. Discussion The proportionate change in median amplitude for the intra-subject comparison (44–53% reduction when further from the lid margin) is broadly similar to that seen in the intergroup comparison (41%), though slightly higher. The ‘‘comfortable’’ position varied substantially in both comparisons (whilst the position 2 mm from the lid margin was consistent), and it is possible that this was more frequently closer to the lid margin in the intergroup comparison than in the intra- subject comparison. This study investigated the effect on the ERG of moving specially designed skin electrodes further inferiorly from the lid margin, both by comparing light-adapted ﬂicker ERGs in two large groups of participants (with the two groups differing by elec- trode position), and by recording light-adapted ﬂash and ﬂicker ERGs in a smaller group in whom recordings were performed using two different posi- tions in consecutive sessions. The ﬁndings from both investigations were consistent: placing the electrode in a more inferior position resulted in a signiﬁcant Previous studies of skin electrode position have shown a reduction in amplitude the further the recording electrode is from the cornea [5, 6, 9]. Also, 123 12 3 3 Doc Ophthalmol Fig. 4 ERG parameters for different electrode positions in participants in the third group (n = 10; data normalised to control eye). a, b, c Boxplots showing a-wave, b-wave and ﬂicker ERG amplitudes: differences were signiﬁcant between the two positions (p = 0.004, 0.002 and 0.002, respectively, Wilcoxon signed rank test). d, e, f, Boxplots showing a-wave, b-wave and ﬂicker ERG peak times: differences were not signiﬁcant (p = 0.85, 0.57 and 0.56, respectively). Boxes show median and upper and lower quartiles; squares plot mean values; whiskers extend to maximum and minimum values. Note that the y-axis scales differ in the lower panels and have been expanded considerably in F, where the actual range between maximum and minimum values is less than 0.1 Fig. 4 ERG parameters for different electrode positions in participants in the third group (n = 10; data normalised to control eye). a, b, c Boxplots showing a-wave, b-wave and ﬂicker ERG amplitudes: differences were signiﬁcant between the two positions (p = 0.004, 0.002 and 0.002, respectively, Wilcoxon signed rank test). d, e, f, Boxplots showing a-wave, b-wave and ﬂicker ERG peak times: differences were not signiﬁcant (p = 0.85, 0.57 and 0.56, respectively). 123 References 1. Maa AY, Feuer WJ, Davis CQ, Pillow EK, Brown TD, Caywood RM, Chasan JE, Fransen SR (2016) A novel device for accurate and efﬁcient testing for vision-threat- ening diabetic retinopathy. J Diabetes Complications 30(3):524–532 Although multiple parameters were compared in the intra-subject comparison, a correction for multiple testing was not deemed necessary [18]. The compar- isons were predetermined and essentially explored differences in only two types of parameter, namely amplitudes and peak times. The ﬁnding that ampli- tudes were signiﬁcantly different, and peak times were not, was observed consistently across all parameter comparisons, strongly supporting the validity of the conclusions of this study. 2. Fukuo M, Kondo M, Hirose A, Fukushima H, Ikesugi K, Sugimoto M, Kato K, Uchigata Y, Kitano S (2016) Screening for diabetic retinopathy using new mydriasis- free, full-ﬁeld ﬂicker ERG recording device. Sci Rep 8(6):36591 3. Nakamura N, Fujinami K, Mizuno Y, Noda T, Tsunoda K (2016) Evaluation of cone function by a handheld non- mydriatic ﬂicker electroretinogram device. Clin Ophthal- mol 30(10):1175–1185 4. Motokawa K, Mita T (1942) Uber eine einfachere Unter- suchungsmethode und Eigenschaften der Aktionsstrome der Netzhaut des Menschen. Tohokku J Exp Med 42:114–133 Acknowledgements AEH is recipient of a College of Optometrists Postgraduate Studentship. OAM receives funding from the Wellcome Trust (Grant 206619/Z/17/Z), Fight for Sight (1409/10), Birdshot Uveitis Society (24BU141), Thomas Pocklington Trust (24TPT16) and the National Institute for Health Research (NIHR) Biomedical Research Centre at Moorﬁelds Eye Hospital and the UCL Institute of Ophthalmology. TwinsUK receives funding from the Wellcome Trust and the NIHR BioResource Clinical Research Facility and Biomedical Research Centre based at Guy’s and St. Thomas’ NHS Foundation Trust and King’s College London, UK. The views expressed are those of the authors and not the funding organisations. The funding organisations had no role in the design or conduct of the research. The authors thank Dr Neringa Jurkute, Dr Sumita Phatak, Dr Peter Heydon, Dr Ahmed Kasb and Ms Priya Francome-Wood for assistance with recordings. 5. Noonan BD, Wilkus RJ, Chatrian GE, Lettich E (1973) The inﬂuence of direction of gaze on the human electroretino- gram recorded from periorbital electrodes: a study utilizing a summating technique. Electroencephalogr Clin Neuro- physiol 35(5):495–502 6. Zhang G, Nao-i N, Honda Y (1983) Topographical distri- bution of electroretinographic potentials monitored by skin- electrodes. J Pediatr Ophthalmol Strabismus 20(4):153–157 7. France TD (1984) Electrophysiologic testing and its speciﬁc application in unsedated children. Discussion Statement of human rights All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Statement on the welfare of animals No animals were used in this research. References Trans Am Ophthalmol Soc 82:383–446 8. Esakowitz L, Kriss A, Shawkat F (1993) A comparison of ﬂash electroretinograms recorded from Burian Allen, JET, C-glide, gold foil, DTL and skin electrodes. Eye (Lond) 7(Pt 1):169–171 9. Kriss A (1994) Skin ERGs: their effectiveness in paediatric visual assessment, confounding factors, and comparison with ERGs recorded using various types of corneal elec- trode. Int J Psychophysiol 16(2–3):137–146 Statement on the welfare of animals No animals were used in this research. 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 unre- stricted 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 Com- mons license, and indicate if changes were made. Also, potential time-dependent adaptational effects (the right eye was recorded from ﬁrst in all subjects) were controlled for, in the ﬁrst two groups, by averaging both eyes, and, in the third group, by normalising the test eye to the control eye. Even when right and left eyes were compared separately in the ﬁrst two groups, the results were found to be similar (amplitudes differed signiﬁcantly between the two positions, but peak times did not). Discussion Boxes show median and upper and lower quartiles; squares plot mean values; whiskers extend to maximum and minimum values. Note that the y-axis scales differ in the lower panels and have been expanded considerably in F, where the actual range between maximum and minimum values is less than 0.1 less critical with respect to analysis of peak time parameters. Nevertheless, the electrode must be posi- tioned sufﬁciently close to the eye to elicit an ERG in which the peaks and troughs are discernible. In this study, all healthy subjects produced a measurable ﬂicker ERG, even when the electrode was positioned up to 20 mm from the lid margin. However, in patients with subnormal responses in retinal disease, this can be more challenging. In some patients with reduced cone system responses measured using conventional ERG recording systems, it has not been possible to accurately determine peak times using the RETeval system as amplitudes are too low to distinguish response components [3]. we have found that ERGs recorded with conductive ﬁbre electrodes placed in the fornix are substantially lower in amplitude than those recorded with the electrode at the lid margin, whilst peak times are similar (Tariq et al. [16], ARVO meeting abstract 5121). This result was consistent with the ﬁndings of a similar study of conductive ﬁbre electrode position recently published, showing a 20–25% reduction in amplitude [17]. Overall, the present study ﬁts well with the ﬁndings of previous studies of both skin and corneal conductive ﬁbre electrodes: the further the recording electrode is from the corneal apex, the lower the recorded signal amplitude. The ﬁndings of the present and previous studies suggest that skin recording electrode positioning is 123 123 Doc Ophthalmol One limitation of the present study was that electrode placement in Group 1 was not consistent and was determined more by participant ‘‘comfort’’, whilst positioning in Group 2 was consistent. Also, the participants were not randomised to different elec- trode positions, but the position was determined by whether they were in the ﬁrst or second phase of subject recruitment. However, recordings from the third group of participants (in whom both positions were used in the same subject) were consistent with the ﬁndings from the ﬁrst two groups. Informed consent All participants gave informed consent. Informed consent All participants gave informed consent. Compliance with ethical standards Conﬂicts of interest The authors declare that they have no conﬂict of interest. 12 123 Doc Ophthalmol contributions to variations in human retinal electrical responses quantiﬁed in a twin study. Ophthalmology 124(8):1175–1185 10. Bradshaw K, Hansen R, Fulton A (2004) Comparison of ERGs recorded with skin and corneal-contact electrodes in normal children and adults. Doc Ophthalmol 109(1):43–55 11. Mortlock KE, Binns AM, Aldebasi YH, North RV (2010) Inter-subject, inter-ocular and inter-session repeatability of the photopic negative response of the electroretinogram recorded using DTL and skin electrodes. Doc Ophthalmol 121(2):123–134 15. Neveu MM, Dangour A, Allen E, Robson AG, Bird AC, Uauy R, Holder GE (2011) Electroretinogram measures in a septuagenarian population. Doc Ophthalmol 123(2):75–81 16. Tariq A, Sheriff I, Bhatti T, Sankoh A, Gao H, Hammond CJ, Mahroo OA (2014) Effect of varying conductive ﬁbre electrode position between fornix and lid margin on elec- troretinogram amplitudes and implicit times. Invest Oph- thalmol Vis Sci 55(13):5121 12. McCulloch DL, Marmor MF, Brigell MG, Hamilton R, Holder GE, Tzekov R, Bach M (2015) ISCEV standard for full-ﬁeld clinical electroretinography (2015 update). Doc Ophthalmol 130(1):1–12. https://doi.org/10.1007/s10633- 014-9473-7 17. Kurtenbach A, Kramer S, Strasser T, Zrenner E, Langrova´ H (2017) The importance of electrode position in visual electrophysiology. Doc Ophthalmol 134(2):129–134 13. Moayyeri A, Hammond CJ, Hart DJ, Spector TD (2013) The UK Adult Twin Registry (TwinsUK Resource). Twin Res Hum Genet 16(1):144–149 18. Armstrong RA (2014) When to use the Bonferroni correc- tion. Ophthalmic Physiol Opt 34(5):502–508 14. Bhatti T, Tariq A, Shen T, Williams KM, Hammond CJ, Mahroo OA (2017) Relative genetic and environmental 123 12
W2497525241.txt	http://sbc.org.pl/Content/511073/PDF/5098.1.pdf	de		Die Schmetterlinge Deutschlands mit besonderer Berücksichtigung ihrer Biologie	null	1,913	public-domain	0

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